a basic introduction to standard molecular biology techniques...in the laboratory practicals, you...

A Basic Introduction To StandardMolecular Biology Techniques

July 26th – August 6th 2010

Course Run by Patricia Dyal

Contact : +44 (0) 207 942 5059/5427Email: [email protected]

Contents Page

Course Timetable

Introduction 1

Laboratory Health and Safety 2

Use of Pipettes 3

Section 1: Isolation of genomic DNA from Cestodes and Digeneans 4

1.1: Tissue solubilisation 5

1.2: Purification of DNA from cell lysate 6

1.3: Agarose gel analysis of purified gDNA 8

1.4: Nanodrop quantitation of DNA 10

Section 2: PCR amplification of a specific genomic region – Large subunit 12

(LSU) and Small subunit (SSU) genes

2.1: PCR reaction setup using RTG beads 13

2.2: Agarose gel analysis of unpurified PCR reactions 15

2.3: Affinity column cleanup of PCR reactions 15

2.4: Agarose gel analysis of purified PCR reactions 17

2.5: Nanodrop quantitation of purified PCR product 17

Section 3: Cycle sequencing 18

3.1: Calculate the amount of DNA needed in sequencing reaction 18

3.2: Sequencing reaction setup 18

Section 4: Isolation of fish genomic DNA using Qiagen DNeasy DNA extraction 20

Kit


4.2: Purification of DNA from cell lysate 21

4.3: Agarose gel analysis of purified genomic DNA 23

4.4: Nanodrop quantitation of purified genomic DNA 23

4.5: PCR amplification of the mitoc hondrial cytochrome b (cytb) and 23

cytochrome oxidase gene (cox1)

Section 5: CTAB genomic DNA Extraction and ethanol precipitation of 26

snail DNA


5.2: Organic extraction 28

5.3: Ethanol precipitation of genomic DNA 29

5.4: Agarose gel analysis of purified genomic DNA 31

Section 6: DNA Gel Extraction 32

6.1 QIAquick Gel Extraction Kit Protocol-using a microcentrifuge 32

6.2: Agarose gel analysis of purified PCR products 35

Section 7: Theoretical Background on Basic Molecular Techniques 36

7.1 DNA extraction 36

7.2 PCR –Principles and Procedures 40

7.3 Agarose Electrophoresis 53

7.4 Purifiction of PCR products 57

7.5 Cycle Sequencing 61

7.6 Sequencher DNA sequencing assembly and analysis software 69

7.7 The basic local alignment search tool (BLAST) 73

Appendix 1: Buffers 74

Appendix 2: Tissue Storage 78

Appendix 3: IUB codes-Nomenclature for Incompletely Specified Bases in 81

Nucleic Acid Sequences

Appendix 4: Consumables and Laboratory equipment 82

Outline Time Table:

Week 1 – 26th-30th July 2010

1. General introduction

Facility

Health & Safety

Training program

2. Set up DNA extractions overnight

3. DNA extractions

4. Gel electrophoresis of extracted DNA

5. PCR set up overnight-Kit based

6. Electrophoresis of PCR products

7. PCR products clean up for Sequencing

8. Quantification of PCR products

9. Set up sequencing reactions overnight.

10. Overview of Sequencing Facility with Julia Llewellyn Hughes (Sequencing facility Manager)

11. Overview of GENIUS –bio-informatics software (Dr. Pete Olson)12. Theoretical overview of all molecular techniques (principles and trouble shooting)

Week 2- 2nd-6th August 2010

1. Meeting Dr. Phil Rainbow – head of Zoology Department

2. Data Analysis

Clean up of sequence data

Types of Sequencing Software

Blast searches

Tree generating software – Dr. Tim Littlewood (3rd August)

3. DNA extraction using CTAB and recovery by organic extractions andethanol precipitation.

4. PCR amplification of Mitochondrial genes using standard PCR reagents

5. DNA Gel extraction – commercial kit based

6. Discussion:

Basic laboratory equipment and consumables required for

setting up a molecular facility.

Storage of specimens for molecular analysis

Storage of nucleic acids, short and long term

7. Meeting with Jackie Mackenzie Dodds (Laboratory Manager to discuss setting up a molecular

collection.

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Introduction

The aim of this molecular training course is to provide initial experience of common

laboratory and computing techniques used for generating and analysing molecular

sequence data:

Extraction and purification of genomic DNA using affinity spin column

Use of agarose gel electophoresis to analyse DNA samples

Amplification of a specific genomic target by the polymerase chain reaction

(PCR)

Purification of PCR products using affinity spin columns

Automated DNA sequencing reactions

DNA sequence editing

DNA sequence identification and analysis using online and local software tools

The theory and practice of phylogenetic reconstruction from aligned sequence

data, and the different inference methods available

In the laboratory practicals, you will be extracting DNA from cestodes (tapeworms) and

digeneans (flukes). This will be a blind study, so from the DNA sequences generated you

should be able to tell which specimens were cestodes or digeneans. If time permits we

will also extract DNA from some fish specimens.

2

Laboratory Health and Safety

Some of the reagents you will be using are toxic and PCR reactions are easily

contaminated with external DNA from the environment, so it is important to wear lab

coats and gloves whenever working at the bench to protect both you and your samples.

Particular hazards will be detailed in the protocol.

Waste solutions from the experimental process should be sealed in the reaction tubes and

stored on your bench for collection and disposal – no liquids from these protocols

should be poured into the sinks unless specifically stated in the protocol.

Empty tubes and discarded pipette tips should be placed in the clear plastic waste bags on

your bench for disposal.

Gloves, paper towels etc should be placed in the yellow sacs at the front of the laboratory.

Wash your hands before leaving the laboratory.

3

Use of pipettors

pipettors are delicate, expensive and liable to contamination

never touch pipettors with bare hands

never touch the barrel of the pipettor

don't drop them onto the bench, place them gently

maximum pipette volume is shown on the thumb knob, minimum volume is 10%

of maximum

never force pipettes beyond their stated volumes

maximum volume minimum volume

P2 2 µl 0.2 µl

P20 20 µl 2 µl

P100 100 µl 10 µl

P200 200 µl 20 µl

P1000 1000 µl 100 µl

always set volumes by adjusting downwards from a larger volume (1/4 turn)

older style models, with the small, round thumb knob, are adjusted using the

internal collar, newer style models with the large, serrated thumb knob can be

adjusted using either the thumb knob or the internal collar

Pipettor settings

P2 P20 P200 P1000

0 2 0 2 0 2 0 1

2 0 2 0 2 0 1 0

0 0 0 0 0 0 0 0

min max min max min max min max

0.2 µl 2 µl 2 µl 20 µl; 20 µl 200 µl 100 µl 1000

µl

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Section 1: Isolation of Cestode And Digenean genomic DNA

Background

In order to isolate DNA from any specimen, you need to first release the DNA from the

cellular compartments that it is held in (nucleus, chloroplast, mitochondrion etc) and then

to separate it away from the other biomolecules present in that extract.

There are a large number of protocols available to isolate either DNA or total nucleic

acid, both "home-made" and commercial. Each technique has both advantages and

disadvantages in terms of speed, complexity, use of toxic chemicals, cost and suitability

to particular groups of organisms. Different types of cellular organisation (prokaryote vs.

eukaryote, animal vs. plant etc.) can require different processing methods and special

techniques may be required for particular taxonomic groups (eg. molluscs).

You will be using a commercial protocol for the isolation of total nucleic acid (genomic

and mitochondrial DNA plus RNA) from general animal tissues (Qiagen’s QIAamp

DNA Mini Kit ). This method uses a pad of DNA-binding matrix supported on a mesh to

trap the nucleic acids as a tissue lysate is spun through it in the presence of a salt buffer.

The filter is then washed in ethanol-based wash buffers which remove any residual lysate

whilst keeping the nucleic acid product bound to the matrix. The matrix is then dried and

the purified, bound nucleic acid is eluted. The process involves 5 basic steps:

Physical, chemical and enzymatic disruption of cellular structure

Binding of DNA and RNA to a affinity column and removal of other cellular

components by centrifugation

Washing the bound nucleic acids by centrifugation

Recovery of nucleic acids from the column by elution and centrifugation

Examination of recovered DNA to assess yield, quality and purity

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1.1 Tissue solublisation

Tissue Samples: Cestodes And Digeneans

1. Otv 6. Nesc

2. Grill 7. Nesc

3. Gsq 8. Otb3

4. Gyrol 9. Elie

5. APHFP 10. Gono

You are each supplied with:

Reagent code

Lysis buffer ATL

Proteinase K (20mg / ml) PK

Ultrapure dH2O in clear 7 ml “bijou” bottle H2O

Cestode or Digenean in ethanol in clear 2 ml screw cap

vial

Unique ID

0.01TE pH 7.5

Consumables

Sterile forceps

1.5 and 2.0 ml tubes

Note, some of the buffers involved in this kit contain chaotrophic salts and must not

be disposed of via the sink (contact with acids liberates cyanide!!!!!!) Therefore keep

all liquid waste materials on the bench and they will be collected from you.

Similarly, all tips and tubes used in this protocol must be placed in the plastic bags

and will be collected for specialist disposal.

1. Make a note of your specimen ID code and label the 1.5 ml tubes with the specimen

ID code.

2. Aliquot 500 µl of 0.01 TE (10mM Tris-HCl pH7.5 / 0.1 mM EDTA) into a 1.5 ml

tube.

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3. Using the forceps remove sample tissue from ethanol and blot dry on a piece of

clean tissue.

4. Cut off approximately 25 mg of tissue with a sterile scalpel blade and place the tissue

in to the TE buffer. Return the remainder of tissue to ethanol.

5. Leave the tissue for a couple of minutes in the TE buffer to allow the ethanol to

diffuse out.

6. Place the following into a 1.5 ml tube:

180 µl of buffer ATL -tissue lysis buffer

20 µl of Proteinase K solution (20 mg / ml stock)

7. Using the forceps, carefully remove the tissue and blot dry on a piece of clean tissue.

Cut up the tissue into small pieces and transfer the to the lysis buffer.

8. Incubate tubes overnight in a the Thermomixer at 56C, mixing at 400rpm.

1.2 Purification of DNA from Cell Lysate

Reagent code

Binding buffer AL

Ultrapure ethanol ETH

Wash buffer 1 AW1

Wash buffer 2 AW2

Elution buffer AE

Consumables

QIAamp spin column

2 ml and 1.5 ml tubes

Note, the buffers contain toxic chaotrophic salts and must not be disposed of via the

sink. Keep all waste materials on the bench and they will be collected.

1. Collect your DNA extraction tube from the thermomixer.

2. Vortex the tubes for 15 seconds and then briefly centrifuge the tubes to remove drops

from inside the lid.

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3. Add 200 µl of Binding Buffer AL to the tube and mix thoroughly by brief vortexing

(15 seconds)

4. Incubate at 70C for 10 minutes. Briefly centrifuge the tubes to remove drops from

inside the lid.

5. Add 200 µl of ethanol (96-100%) to the tube and mix thoroughly by brief vortexing

(15 seconds). Briefly centrifuge the tubes to remove drops from inside the lid.

It is essential that the sample, Buffer AL, and the ethanol are mixed thoroughly to yield a

homogeneous solution. A white precipitate may form on addition of ethanol. It is essential

to apply all of the precipitate to the QIAamp Mini spin column. This precipitate does not

interfere with the QIAamp procedure or with any subsequent application.

6. Unwrap the spin column / tube and pipette the mixture from step 5 into the top of the

column

7. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute (ensure

that the rotor is balanced before running the microfuge)

8. Transfer the column to a clean 2 ml collection tube

9. Add 500 µl of Wash Buffer AW1 to the spin column



11. Transfer the column to a clean 2 ml tube and add 500 µl of Wash Buffer AW2 to the

spin column

12. Place the column / tube into a microfuge and spin at 14000 rpm for 3 minutes.

13. Transfer the column to a clean 2 ml tube and cut off the cap and hinge

14. Place the column / tube into a microfuge and spin at 14000 rpm for 1 minute . This

dry spin ensures that the membrane is completely dry (elution of DNA from the

membrane will fail if it retains any of the ethanol-containing wash buffer)

15. Transfer the column to a clean 2.0 ml tube and cut off the cap and hinge

16. Pipette 100 µl of Elution buffer Buffer AE directly onto the membrane in the spin

column (take care to not touch / puncture the membrane with the pipette tip) and

leave for 5 minutes.

17. Place the column / tube into a microfuge and spin at 8000 rpm for 1 minute.

8

18. Leaving the spin column in the same tube, pipette a further 100 µl of Elution buffer

Buffer AE directly onto the membrane in the spin column (take care to not touch /

puncture the membrane with the pipette tip) and leave for 5 minutes.


20. Remove and discard the spin column, and transfer the eluted, purified DNA

(approximately 200 µl) to a labelled, capped 1.5 ml tube.

Note: Never vortex the eluted genomic DNA as this will fragment the high molecular

weight DNA, just flick the tube to mix the DNA, spin briefly

1.3 Agarose Gel Analysis of Purified DNA

Before you use a purified DNA extract, you need to confirm the success of the extraction

(have you got any?), assess the quality of the extracted DNA (is it intact or broken up?)

and determine its concentration (how much have I got?). This is initially done by running

out a sample of your extracted DNA on an agarose gel, together with a reference DNA

ladder containing DNA fragments of known size and quantity. Cross referencing of your

sample to the bands of the ladder enables the size, quality and yield of your sample to be

assessed.

1.3.1 Making the gel (for a 1% gel, 100mL volume)

1. Weigh out 1.0 of agarose into a 250 mL conical flask. Add 100ml of 1 x TAE, swirl

to mix. It is god to use a large container, as long as it fits n the microwave, because

the agarose boils over easily.

2. Microwave for about 3-4 minutes to dissolve the agarose. The agarose solution can

boil over very easily so keep checking it. It is good to stop it after 45 seconds and give

it a swirl. It can become superheated and NOT boil until you take it out whereupon it

boils out all over you hands. So wear gloves and hold it at arms length.

3. Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep

holding in bare hands). If you had to boil it for a long time to dissolve the agarose

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then you may have lost some water to water-vapour. You can weigh the flask before

and after heating and add in a little distilled water to make up this lost volume. While

the agarose is cooling, prepare the gel tank ready, on a level surface.

4. Add 10 µl of Gel Red (5 µl per 50 ml) and swirl to mix. Pour the gel slowly into the

tank. Push any bubbles away to the side using a disposable tip. Insert the comb and

double check that it is correctly positioned.

5. Leave to set for at least 30 minutes, preferably 1 hour. The gel may look set much

sooner but running DNA into a gel too soon can give terrible-looking results with

smeary diffuse bands.

6. Pour 1 x TAE buffer into the gel tank to submerge the gel to 2–5mm depth. This is the

running buffer.

1.3.2 Preparing the samples

7. Into a clean 0.5 ml centrifuge tube place the following and mix by gentle aspiration:

3 µl of your DNA sample

2 µl of loading dye

8. Write in your lab-book the physical order of the tubes so you can identify the lanes on

the gel photograph.

9. Load the first well with 5 µl of the Hyperladder 1 size marker. Continue loading the

samples and finish of with a final lane of marker.

I load gels from right to left with the wells facing me. This is because gels are published,

by convention, as if the wells were at the top and the DNA had run down the page. If this

seems confusing then you can load left to right with the wells facing away from you.

10. Close the gel, tank switch on the power source and run the gel at 5V/cm.

For example, if the electrodes are 10cm apart then run the gel at 50V. It is fine to run the

gel slower than this but do not run any faster. Above 5V/cm the agarose may heat up and

begin to melt with disastrous effects on your gel's resolution. Some people run the gel

slowly at first (eg. 2V/cm for 10 minutes) to allow the DNA to move into the gel slowly

and evenly, and then speed up the gel later. This may give better resolution. It is OK to

run gels overnight at very low voltages, eg. 0.25–0.5V/cm. Check that a current is

10

flowing. You can check this on the power-source, the milliamps should be in the same

ball-park as the voltage, but the best way is to look at the electrodes and check that they

are evolving gas (ie. bubbles). If not then check the connections, that the power-source is

plugged in etc.

Monitor the progress of the gel by reference to the marker dye. Stop the gel when the

bromophenol blue has run 3/4 the length of the gel. Switch off and unplug the gel tank

and carry the gel (in its holder if possible) to the dark-room to look at on the UV light-

box and photograph. Note: UV is carcinogenic and must not be allowed to shine on

naked skin or eyes. So wear face protection, gloves and long sleeves.

1.4 Nanodrop Quantification of DNA

Before proceeding further into costly and time-consuming manipulations it is critical to

analyze, at least in a cursory way, the quantity and quality of DNA in the prep. A more

accurate quantitation of your sample can be acheived using a "Nanodrop" UV

spectrophotometer .The machine's use will be demonstrated to you. Only ever use non-

scratching, non linting-tissues to clean the instrument.

1. Switch on machine

2. Start up the Nanodrop software and login. Select the "nucleic acid" option

3. Follow the onscreen instructions - place 1.5 µl of ultrapure water onto the sample

pedestal, close the arm and click on OK to initialise the instrument.

4. Once the instrument has initialised, lift the arm and carefully wipe the sample

pedestal and arm Perform a blanking reaction using 1.5 µl of whatever your sample is

dissolved in (in our case, buffer AE), close down the arm and click on the blank

button

5. Once the instrument has blanked (graph with zeroed baseline), lift the arm and

carefully wipe the sample pedestal and arm

6. Perform a measurement - place 1.5 µl of your sample onto the sample pedestal, name

it in the sample ID window, close down the arm and click on the measure button.

Note: It is advisable to blank the Nano drop after every sixth sample

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7. Record the concentration (ng / µl) and the OD 260/280 ratio:

Estimating DNA Concentration by A260

The UV absorbance spectrum of DNA exhibits an Amax @ 260 nm based on the

aromatic ring structures of the DNA bases. This is the most convenient way to estimate

DNA concentration and calculate yield, as long as the DNA preparation is relatively free

of contaminants that absorb in the UV. Proteins, and residual phenol left from the

isolation procedure, are typical contaminants that may lead to an overestimate DNA

concentration.

An A260 = 1.0 indicates a [DNA] = 50 ug/ul, assuming the DNA is pure.

Single-stranded DNA: 33 ug/ul

RNA: 40 ug/ul

260/280: The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of

DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of

~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either

case, it may indicate the presence of protein, phenol or other contaminants that absorb

strongly at or near 280 nm.

260/230 Ratio: This ratio is used as a secondary measure of nucleic acid purity. The

260/230 values for “pure” nucleic acid are often higher than the respective 260/280

values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is

appreciably lower than expected, it may indicate the presence of contaminants which

absorb at 230 nm

12

Section 2: PCR Amplification of A Specific Genomic Region–

Large subunit (LSU) and Small subunit (SSU) genes

Background

You will be amplifying a selected target region of the nuclear 18S (SSU) and 28S (LSU)

gene for the cestodes and digeneans. Subsequently, you will use the PCR product as

template in sequencing. For a PCR reaction, we need to combine, in the correct relative

amounts:

DNA template

the 2 target-specific primer

buffer

dNTPs

Taq polymerase

To set up a PCR reaction it is possible to individually add each of these components.

However, for simplicity, and standardisation, you will be using llustra PuReTaq Ready-

To-Go PCR Beads Catlog no. 27-9559-01 (GE-Healthcare). These are commercial

preparations of dNTPs, buffer and Taq polymerase, that have been mixed together,

dispensed in the correct amounts for a single 25µl PCR reaction and then lyophilised into

a small bead that is stable for many months at room temperature. The additional

advantage of using PCR beads is that they minimise the number of stock solutions and

pipetting steps required for PCR reaction setup, thereby also minimising the risk of

contaminating the reaction with DNA from external sources. This is especially

important when using "universal" primers as they will also potentially amplify the

gene from any contaminating DNA and PCR is a very sensitive process. Liquid PCR

premixes are also available, some also include a gel loading dye so you can pipette

straight from the finished PCR reaction onto a gel.

13

PCR and Sequencing Primers

2.1 PCR Reaction Set up using RTG Beads

When setting up the reactions, use a new pipette tip for every pipetting step, pipette

into the bottom of the tubes on the side without touching the bead and avoid

touching the inside face of the cap.

1. Label the PCR bead tubes with sample ID and label one tube as the negative control

tube. Write on the top of the side wall, not on the tube cap as this can get rubbed

off.

Always include a negative and a positive control when setting up your PCR. The negative

control will give an indication of any reagent contamination. A positive control will

confirm your PCR conditions are working.

2. To each label tube add the follwing:

primer P1 10 M) 1 µl

primer P2 (10M) 1 µl

Sterile water 22 µl

DNA 1 µl

Note: If you are setting up several reactions using the same primer pair, it is best to

prepare a master mix of the primers and water. This will reduce the number of pipetting

SSU-18S LSU-28S

PCR Primers Worm A + Worm B LSU-5’F + 1500R

Sequencing Primers: 300F ECD2S

930F 500F

1200F 900F

1270F 1200F

600R 300R

1200R 400R

1270R 1200R

PCR product size 1700 bp 1500 bp

14

steps and the risk of contamination. For Example: I need to set up 10 reactions

including a negative reagent control.

X-1 X-10

primer P1 10 M) 1 µl 10 µl

primer P2 (10M) 1 µl 10 µl

DNA 1 µl -

Sterile water 22 µl 220 µl

25µl 240 µl

Aliquot 24 µl of the master mix to your label tubes, then add a 1 µl of your respective

template.

3. Vortex the tubes and spin briefly to bring the contents to the bottom of the tube.

Once all the PCR reactions are set up, they will be cycled overnight under the following

conditions:

LSU -PCR Cycling Conditions

Step Temperature (C) Duration Description #1 95 5 min Initial

denaturationx 1

2 95 30 sec Denaturation

3 55 30 sec Annealing x 404 72 2 min Extension

5 72 10 min Final extension x 16 4 hold x 1

SSU -PCR Cycling Conditions

Step Temperature (C) Duration Description #

1 95 5 min Initialdenaturation

x 1

2 94 30 sec Denaturation

3 54 30 sec Annealing x 354 72 2 min Extension


PCR reactions can be stored at -20oC until purification.

15

2.2 Agarose Gel Analysis of Un-purified PCR Reactions

1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3.1

2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle aspiration:

3 µl of your PCR sample


3. Carefully load your 5 µl sample of (PCR reaction plus loading dye) onto the agarose

gel (note the order in which you load your samples).

4. In a separate lane load 5 µl of the molecular standard, Hyperladder 1, which allows

you to determine the size of your PCR product.

5. The gel will be run at 60V for 45 minutes, examined and photographed under UV.

2.3 Affinity Column Cleanup of PCR reactions (Qiagen QIA quick

PCR Purification Kit)

Before the PCR product can be used in a DNA sequencing reaction, it must be purified

from the other components of the PCR reaction mix (especially any unincorporated PCR

primers, which would otherwise act as initiation points for additional, unwanted

sequencing reactions and so give multiple, overlaying, sequencing reads from the same

sequencing reaction). There are many techniques available to purify PCR products. Here

we will be using a commercial affinity column method (Qiagen QIA quick Spin PCR

cleanup columns). Like the QIAamp protocol, it uses a pad of DNA-binding matrix

supported on a mesh to trap the PCR product as the PCR reaction is spun through it in the

presence of a high salt buffer. The filter is then washed in an ethanol-based solution

which removes any residual reaction components whilst keeping the PCR product bound

to the matrix. The matrix is then dried and the purified, bound PCR product then eluted

using a small volume of low salt buffer or water. However the column and buffers differ

in formulation between the 2 kits.

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Purify the amplified DNA from the remaining 22 µl of your PCR reaction mix using the

following protocol (note: do not purify the negative control which can now be

discarded)

Reagent code

QiaQuick spin column in 2 ml centrifuge tube

Buffer PB (DNA binding buffer) PB

Buffer PE (wash buffer) PE

Buffer EB (elution buffer, (10 mM Tris·Cl, pH

8.5))

EB


sink. Keep all waste materials on the bench and they will be collected for specialist

disposal.

1. To the remaining 22 µl of PCR reaction add 110 µl ( = 5 volumes) of Buffer PB and

mix well by brief vortexing (2 seconds).

2. Apply the entire sample to the QIAquick column

3. Place the column / tube into a microfuge and spin at full speed (13000 rpm) for 1

minute (ensure that the rotor is balanced before running the microfuge)

4. Transfer the QIAquick column to a clean 2 ml tube and cut off the cap and hinge

5. Add 750 µl of buffer PE to the QIAquick column

6. Place the column / tube into a microfuge and spin at full speed for 1 minute (ensure




that the rotor is balanced before running the microfuge) – this dry spin ensures

that the membrane is completely dry (elution of PCR product from the membrane will

fail if it retains any of the ethanol-containing wash buffer)


17

10. Pipette 30 µl of Elution Buffer EB directly onto the membrane in the spin column

(take care to not touch / puncture the membrane with the pipette tip) and leave

for 1 minute

Note: it is important that the elution buffer completely soaks into the membrane



12. Remove and discard the spin column, and transfer the eluted, purified PCR product

(approximately 28 µl) to a labelled, capped 1.5 ml tube

2.4 Agarose gel analysis of purified PCR Products

1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3

2. Into a clean, labelled 0.5 ml centrifuge tube, place the following and mix by gentle

aspiration:

3 µl of your purified PCR sample


3. Carefully load your 5 µl sample of (purified PCR product plus loading dye) onto the

agarose gel (note the order in which you load your samples).

4. In a separate lane load 5 µl of the molecular standard, Hyperladder 1, which allows

you to determine the size of your PCR product.

5. The gel will be run at 60V for 45 minutes, examined and photographed under UV

6. After loading the gel, place the tube containing the remainder of your purified PCR

reaction (approx 22 µl) on ice.

2.5 Nanodrop quantitation of purified PCR product

Accurately quantify your purified PCR product using the "Nanodrop" UV

spectrophotometer as in section 1.4

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Section 3: Cycle Sequencing

Background

Sequencing both strands of the sequence of interest is highly recommended as it provides

a double check for accuracy - data from each strand acts to verifying that from the other.

Since (a) your PCR product is 1500 (28S) & 1700 base pairs long (depending on the

source species) and (b) a single sequencing reaction is unlikely to read more than 800

bases at highest quality, we need to perform several sequencing reactions in both

directions to obtain a the whole double stranded sequence.

3.1. Calculate the amount of DNA needed in sequencing reaction

Need 2 ng of DNA for every 100bp of product

e.g. Quantified 18S PCR product is 18 ng/µl

18S PCR product is 700 bp long

Therefore need 14 ng (2 x 7) of PCR product in the sequencing reaction

# ng needed/ # ng per ul quantified = # µl PCR product per 10 µl seq rxn

14 ng / 18 ng / µl = 0.78 µl

3.2 Setting Up the Sequencing reaction

1. Remove the dilution buffer and big dye from the freezer and defrost on ice.

2. Mix the contents of each tube by vortexing and then spin tubes to remove contents

from the lids. Keep tubes on ice .

3. Prepare your primer stock; this should be at 1 pmol/µl.

4. Switch on the PCR machine to warm up.

5. Note: Your total sequence reaction volume is 10 µl.

Set up your sequencing reaction in 0.2 ml tubes as follows on ice: -

DNA (PCR product) 0.78 µl

Primer (1 pmol/ul) 1.0 µl

Big dye dilution buffer from kit (2.5x) 2.0 µl

Sterile water 4.22 µl

Total 8.0 µl

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General note:

If you are using the same primer for a number of templates you can prepare a master mix

of primer and dilution buffer.

e.g. 10 reactions using SSU 5’F primer.

Master mix:- SSU 5’F 1 pmol/µl 10 µl

Big dye dilution buffer (2.5x) 20 µl

Total 30 µl

Then aliquot 3.0 µl of the master mix per tube. This saves time on pipetting and reduces

the risk of cross contamination.

6. Mix the contents of your tube by gentle pipetting, if there are air bubbles or liquid on

the side of the tube then give the tube a quick spin in the centrifuge. Return tube to ice.

8. Transfer tubes to PCR machine, select program call Big dye terminator.

9. Once the hot start section of the program is completed (5 min, pause 2 sec before this

cycle is complete).

10. Transfer tube to ice, add 1 µl of dilution buffer and 1.0 µl of Big Dye terminator mix.

Mix by gently pipetting up and down. Note: your total reaction volume is now 10 µl.

Note: If you are processing a large number of samples, you can prepare a master

mix of the Big dye and dilution buffer. Aliquot 2.0 µl of the master mix per tube, this

increases pipetting accuracy and saves time on pipetting.

11. After the addition of the big dye return the tube to PCR machine and press resume,

this will continue the program.

PCR Profile:

96˚C 5 min Hot start

96˚C 10 sec

50˚C 05sec 28 cycles

60˚C 4 min

The PCR should take about 2.30 hours to complete.

20

Section 4: Isolation of fish genomic DNA using Qiagen DNeasyDNA extraction Kit

You will be using a commercial protocol for the isolation of total nucleic acid (genomicand mitochondrial DNA plus RNA) from general animal tissues (Qiagen DNeasy Blood& Tisue Kit).

Tissue Samples:

1. LR3754 = Channa gachua2. LR3731 = Luciocephalus pulcher

4.1 Tissue Solublisation


Reagent code

Lysis buffer ATL


Ultrapure dH2O in clear 7 ml “bijou” bottle H2O

Cestode or Digenean in ethanol in clear 2 ml screw cap

vial

Unique ID

0.01TE pH 7.5

Consumables

Sterile forceps

1.5 and 2.0 ml tubes

Note, some of the buffers involved in this kit contain chaotrophic salts and must not

be disposed of via the sink (contact with acids liberates cyanide!!!!!!) Therefore keep

all liquid waste materials on the bench and they will be collected from you.

Similarly, all tips and tubes used in this protocol must be placed in the plastic bags

and will be collected for specialist disposal.

9. Make a note of your specimen ID code and label the 1.5 ml tubes with the specimen

ID code.

10. Aliquot 500 µl of 0.01 TE (10mM Tris-HCl pH7.5 / 0.1 mM EDTA) into a 1.5 ml

tube.

21

11. Using the forceps remove sample tissue from ethanol and blot dry on a piece of

clean tissue.

12. Cut off approximately 25 mg of tissue with a sterile scalpel blade and place the tissue

in to the TE buffer. Return the remainder of tissue to ethanol.

13. Leave the tissue for a couple of minutes in the TE buffer to allow the ethanol to

diffuse out.

14. Place the following into a 1.5 ml tube:

180 µl of buffer ATL -tissue lysis buffer

20 µl of Proteinase K solution (20 mg / ml stock)

15. Using the forceps, carefully remove the tissue and blot dry on a piece of clean tissue.

Cut up the tissue into small pieces and transfer the to the lysis buffer.

16. Incubate tubes overnight in a the Thermomixer at 56C, mixing at 400rpm.

4.2 Purification of DNA from Cell Lysate

Reagent code

Binding buffer AL

Ultrapure ethanol ETH

Wash buffer 1 AW1

Wash buffer 2 AW2

Elution buffer AE

Consumables

DNeasy spin column

2 ml and 1.5 ml tubes


sink. Keep all waste materials on the bench and they will be collected.

21. Collect your DNA extraction tube from the thermomixer.

22. Vortex the tubes for 15 seconds and then briefly centrifuge the tubes to remove drops

from inside the lid.

22

23. Add 200 µl of Binding Buffer AL to the tube and mix thoroughly by brief vortexing

(15 seconds)

24. Add 200 µl of ethanol (96-100%) to the tube and mix thoroughly by brief vortexing

(15 seconds). Briefly centrifuge the tubes to remove drops from inside the lid.

It is essential that the sample, Buffer AL, and the ethanol are mixed thoroughly to yield a

homogeneous solution. A white precipitate may form on addition of ethanol. It is essential

to apply all of the precipitate to the DNeasy Mini spin column. This precipitate does not

interfere with the DNeasy procedure or with any subsequent application.

25. Unwrap the spin column / tube and pipette the mixture from step 5 into the top of the

column



27. Transfer the column to a clean 2 ml collection tube

28. Add 500 µl of Wash Buffer AW1 to the spin column



30. Transfer the column to a clean 2 ml tube and add 500 µl of Wash Buffer AW2 to the

spin column

31. Place the column / tube into a microfuge and spin at 14000 rpm for 3 minutes.


33. Place the column / tube into a microfuge and spin at 14000 rpm for 1 minute . This

dry spin ensures that the membrane is completely dry (elution of DNA from the

membrane will fail if it retains any of the ethanol-containing wash buffer)

34. Transfer the column to a clean 2.0 ml tube and cut off the cap and hinge

35. Pipette 100 µl of Elution buffer Buffer AE directly onto the membrane in the spin

column (take care to not touch / puncture the membrane with the pipette tip) and

leave for 5 minutes.


37. Leaving the spin column in the same tube, pipette a further 100 µl of Elution buffer

Buffer AE directly onto the membrane in the spin column (take care to not touch /

puncture the membrane with the pipette tip) and leave for 5 minutes.

23


39. Remove and discard the spin column, and transfer the eluted, purified DNA

(approximately 200 µl) to a labelled, capped 1.5 ml tube.

4.3 Agarose gel analysis of purified genomic DNA


2. Into a clean 0.5 ml centrifuge tube, place the following and mix by gentle

aspiration:

3. 3 µl of your genomic DNA

4. 2 µl of loading dye

5. Carefully load your 5 µl sample of (DNA plus loading dye) onto the agarose gel

(note the order in which you load your samples).

6. In a separate lane load 5 µl of the molecular standard, Hyperladder 1, which

allows you to determine the size of your DNA.


4.4 Nanodrop quantitation of purified genomic DNA

Accurately quantify your purified genomic DNA using the "Nanodrop" UV

spectrophotometer as in section 1.4

4.5 PCR amplification of the mitochondrial cytochrome b (cytb) and

cytochrome oxidase (cox1) genes

PCR and Sequencing Primers

DNA Samples

1. LR3752 = Channa melasoma2. LR3754 = Channa gachua

cytb CO1PCR Primers Part1-LRP211 + LRP21 Forward LRP140 + LRP142

PCR product size part 1 and 2 = 700 bp cox1 = 650 bp

24

3. LR3731 = Luciocephalus pulcher

PCR Protocol

1. Defrost all PCR reagents, except the Taq Polymerase which is kept on ice.

2. Once thawed, vortex tubes and spin briefly. Place all tubes on ice.

3. Label the PCR tubes with sample ID and label one tube as the negative control tube.

Write on the top of the side wall, not on the tube cap as this can get rubbed off.

Always include a negative and a positive control when setting up your PCR. The negative

control will give an indication of any reagent contamination. A positive control will

confirm your PCR conditions are working.

4. To each label tube add the PCR components in the following order:

Sterile water 13.8 ul

5 X Go Taq Flexi buffer 5 ul

25 mM Magnesium chloride 3 ul

10 uM dNTPs 1 ul

Primer 1 0.5 ul

Primer 2 0.5 ul

Go Taq 5U/ul 0.2 ul

DNA 1 ul

25 ul

5. Vortex the tubes and spin briefly to bring the contents to the bottom of the tube.

Once all the PCR reactions are set up, they will be cycled overnight under the following

conditions:

PCR Cycling Conditions

25

Step Temperature (C) Duration Description #

1 94 3 min Initialdenaturation

x 1

2 94 1 min Denaturation

3 52-54 1 min Annealing x 354 72 1 min 30

secExtension


6. Run 3 ul of your PCR reaction on a 1% gel in TAE as in section 2.4.

26

Section 5: CTAB genomic DNA Extraction and ethanol

precipitation of snail DNA

Background

Certain tissue types and organisms provide more of a challenge in terms of DNA

extraction, with the "quick and easy" kit based techniques producing poor yields or

damaged (sheared) DNA. Snails can be problematic, the actual tissue is tough and

rubbery, so it is hard to physically break down and digest, and conventional techniques

may shear the DNA. An alternative, "home brew" technique can produce better results.

You will use a CTAB extraction technique coupled with organic extraction and ethanol

precipitation to extract DNA from a specimen of Biomphalaria glabrata.

CTAB (Cetyl Trimethyl Ammonium Bromide) is a powerful cationic surfactant

which helps disrupt and lyse the snail tissue. Proteinase K is also used to break down

proteins.

Organic compounds such as phenol and "chloroform" (see below for explanation for

the quote marks) are immiscible with aqueous solutions, yet denature and precipitate

out proteins from them, whilst leaving nucleic acids untouched; different subsets of

proteins are precipitated by the different organic compounds. Therefore mixing these

compounds with the snail lysate and then separating the aqueous and organic phases

by brief centrifugation (the aqueous phase is the top (lighter) one) clears the aqueous

phase of proteins, which either form a pellet at the bottom of the tube or a layer at the

interface between the phases. Because pure chloroform can be partly miscible with

water, it is always used as a 24:1 mix with isoamyl alcohol, which allows for a clearer

interface between the organic and aqueous phases. Hence "chloroform" refers to this

24:1 mix. Extraction can either be undertaken sequentially with phenol, then with a

1:1 mix of phenol-"chloroform", and finally with "chloroform", else 2 rounds of

extraction with a 1:1 mix of phenol-"chloroform" can be performed

In order to obtain DNA (actually total nucleic acids) from the resulting protein-free

lysate, it s precipitated out of solution with alcohol and recovered by centrifugation.

The DNA pellet is then washed, dried and redissolved in an appropriate aqueous

buffer. Nucleic acids precipitate out of aqueous solution in the presence of >67%

ethanol or >50% isopropanol. Either can be used, one the one hand, ethanol tends to

27

produce a slihghtly milky pellet whilst that with isopropanol is more glassy and so is

harder to see, on the other hand, the total sample volume is larger with ethanol

(volume of aqueous sample plus 2 volumes of 100% ethanol) than with isopropanol

(volume of aqueous sample plus 1 volume of 100% isopropanol) and so, depending

on sample volume, it may need to be divided between 2 tubes for the precipitation

step (so mote handling time). Often salts are added to the aqueous phase to promote

precipitation (eg ammonium acetate, sodium acetate, sodium chloride), hence the

pellet-rinsing step, to eliminate salt carry over into the purified DNA solution.

Note: both phenol and "chloroform" are toxic - standard laboratory schemes of

work dictate that these steps are conducted in a fume hood. Because you will be

using small volumes that have been pre-aliquoted for you, we have assessed that it is

safe to conduct this practical exercise on the open bench. If you wish to use a fume

cupboard, you are welcome to do so. All tips that are used during the organic

extraction step must be kept separate for specialist disposal, and the organic phases

must be sealed in tubes immediately after use, labelled and left on the bench for us

to collect.

5.1 Tissue solublisation


Reagent Code

"x2" CTAB lysis buffer CTAB


Frozen snail tissue

Consumables

Plastic petri dish

Plastic microfuge pestle

Screw cap 1.5 ml tube

1. Label the 1.5 ml screw cap tube.

28

2. Fresh or alcohol preserved animal tissue should be damp-dried quickly using a

kimwipe, diced into small pieces using a scalpel blade.

3. Transfer the macerated tissue to the 1.5 ml screw cap tube and label it with your

name

4. To the tube add 380 µl of CTAB extraction buffer and 20 µl of proteinase (20mg/ml

stock solution).

5. Crush the tissue well with a microfuge pestle.

6. Incubate in the rotating incubator at 55C overnight

5.2 Organic Extraction


Reagent Code

Phenol:"chloroform" PC

Chloroform:isoamylalcohol (24:1)

Consumables

1.5 ml microfuge tubes

1. Transfer the snail lysate to a 1.5 ml microfuge tube

2. Place the tube into a microfuge and spin at full speed for 1 minute (ensure that the

rotor is balanced before running the microfuge) to pellet residual undigested

tissue.

3. Transfer the supernatant to a clean 1.5 ml tube, add 400 µl of phenol / "chloroform"

and firmly cap the tube

4. Gently invert the tube 20 times to form an emulsion.

5. Place the tube into a microfuge and spin at full speed for 3 minutes (ensure that the

rotor is balanced before running the microfuge).

6. Transfer the aqueous (top) phase to a clean, labelled 1.5 ml tube. Try not to transfer

any of the precipitated protein from the interface. It is better to leave a small

amount of the aqueous phase behind. Cap the waste tube containing the organic

phase.

29

7. Add 400 µl chloroform / isoamyl alcohol (24:1) to the aqueous phase and firmly cap

the tube

8. Gently invert the tube 20 times to form an emulsion.


rotor is balanced before running the microfuge).

10. Transfer the aqueous (top) phase to a clean, labelled 1.5 ml tube. It is essential to not

transfer any precipitated protein from the interface, or chloroform. Cap the

waste tube containing the organic phase.

5.3 Ethanol Precipitation of genomic DNA


Reagent Code

10M sodium acetate SA

100% ultrapure ethanol Eth

70% ultrapure ethanol 70

Ultrapure water H2O

Consumables

Disposable plastic pasteur pipettes

Plastic petri dish

1.5 ml microfuge tubes

1. Add 40 µl of 10M sodium acetate (one tenth of the volume) to the final aqueous

phase from the organic extraction.

2. Add 880 µl of 100% ethanol (ie. 2 x the combined volume of the original aqueous

phase plus the acetate).

3. Vortex briefly to mix and then place in -70oC freezer for 5 minutes.

4. Remove tube from freezer and allow to warm up on bench for a couple of minutes (if

it goes straight into the centrifuge from the freezer, the tube will be brittle and

may shatter).

30


rotor is balanced before running the microfuge and that you place the tube into

the centrifuge with its hinge pointing outwards).

6. Carefully remove the supernatant with a plastic pasteur pipette and discard into the

petri dish (nb. The pellet may not be visible, but it will be on the side wall of the

tube down from the cap hinge, slide the tip of the pipette slowly down the

opposite wall of the tube, gently sucking up the liquid as you go).

7. Wash the pellet by adding 1000 µl of 70% ethanol and vortexing briefly.

8. Re-pellet the DNA by spinning at 14000 rpm for 2.5 minutes (ensure that the rotor

is balanced before running the microfuge and that you place the tube into the

centrifuge with its hinge pointing outwards).

9. Carefully remove and discard the supernatant (as above).

10. Spin the tube again for a few seconds (ensure that the rotor is balanced before

running the microfuge and that you place the tube into the centrifuge with its

hinge pointing outwards) and remove the last of the liquid.

11. Place the tube, with its cap off, in the heat block at 37oC for about 5 minutes to dry

the pellet / evaporate any remaining drops of fluid (do not overdry the pellet - check

it regularly and remove the tube as soon as there is no fluid left).

12. Once the pellet is dry, add 50 µl of ultra water and incubate in the heat block at 37oC

for 30 minutes to redissolve the DNA. On 3- 4 ocassions during this period, gently

tap the side of the tube to aggitate its contents.

13. Store extracted DNA at -20oC

Note:

samples can be redissolved overnight at 4oC as an alternative.

Redissolving DNA in water, and storing at 4 oC is fine for short term storage (a few

weeks maximum). For longer term storage, samples should be redissolved in TE

buffer (10 mM Tris, 1 mM EDTA) and stored at -20oC. For most purposes, repeated

freeze-thawing will not harm the DNA.

31

Agarose gel analysis of purified gDNA


Reagent code

Loading dye LD

Hyperladder 1

1. Prepare agarose gel as in section 1.3.

2. Into a clean 0.5 ml centrifuge tube place the following and mix by gentle

aspiration:

3. 3 µl of your DNA sample


5. Carefully load your 5 µl sample of (DNA plus loading dye) onto the agarose gel

(note the order in which you load your samples).


allows you to determine the size of your DNA.


32

Section 6: DNA Gel Extraction

DNA gel extraction is used to purify DNA from contaminants or to purify a specific

fragment from a PCR reaction that has several fragments.

6.1 Agarose gel electrphoresis of PCR product

1. Prepare a 1% agarose gel in 1 x TAE as in section 1.3.

Note use the wide combs when making the agarose gel if the bands are going to

be excised.


aspiration:

3. 22 µl of your PCR sample (PCR sample remaining)


5. Carefully load your 27 µl sample of (PCR reaction plus loading dye) onto the



allows you to determine the size of your PCR product.

7. Run the gel at 60V until it has run almost to the end, examined and proceed tosection 6.2 below.

6.2 QIAquick Gel Extraction Kit Protocol-using a microcentrifuge

This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or

low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per

spin column.

Notes: • The yellow color of Buffer QG indicates a pH ≤7.5.

• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

• Isopropanol (100%) and a heating block or water bath at 50°C are required.

• All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a

conventional table-top microcentrifuge.

• 3 M sodium acetate, pH 5.0, may be necessary.

33

1. View the agarose gel on a longwave UV light box. Excise the DNA fragment

from the agarose gel with a clean, sharp scalpel.

Minimize the size of the gel slice by removing extra agarose.

2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1

volume of gel (100 mg ~ 100 μl).

For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose

gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick

column is 400 mg; for gel slices >400 mg use more than one QIAquick column.

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved).

To help dissolve gel, mix by vortexing the tube every 2–3 min during the

incubation.

IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation

time.

4. After the gel slice has dissolved completely, check that the color of the

mixture is yellow (similar to Buffer QG without dissolved agarose).

If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate,

pH 5.0, and mix. The color of the mixture will turn to yellow.The adsorption of

DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains

a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH,

allowing easy determination of the optimal pH for DNA binding.

5. Add 1 gel volume of isopropanol to the sample and mix.

For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. This step

increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments

between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not

centrifuge the sample at this stage.

6. Place a QIAquick spin column in a provided 2 ml collection tube.

7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for

1 min.

The maximum volume of the column reservoir is 800 μl. For sample volumes of

more than 800 μl, simply load and spin again.

8. Discard flow-through and place QIAquick column back in the same

collection tube. Collection tubes are re-used to reduce plastic waste.

34

9. (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge

for 1 min.

This step will remove all traces of agarose. It is only required when the DNA will

subsequently be used for direct sequencing, in vitro transcription or

microinjection.

10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1

min.

Note: If the DNA will be used for salt sensitive applications, such as blunt-end

ligation and direct sequencing, let the column stand 2–5 min after addition of

Buffer PE, before centrifuging.

11. Discard the flow-through and centrifuge the QIAquick column for an

additional 1 min at 13,000 rpm (~17,900 x g).

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed

unless the flow-through is discarded before this additional centrifugation.

12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

13. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the

center of the QIAquick membrane and centrifuge the column for 1 min.

Alternatively, for increased DNA concentration, add 30 μl elution buffer to

the center of the QIAquick membrane, let the column stand for 1 min, and

then centrifuge for 1 min.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the

QIAquick membrane for complete elution of bound DNA. The average eluate

volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl. Elution

efficiency is dependent on pH. The maximum elution efficiency is achieved

between pH 7.0 and 8.5. When using water, make sure that the pH value is within

this range, and store DNA at –20°C as DNA may degrade in the absence of a

buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM

EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions

6.2 Agarose gel analysis of PCR product

35


Reagent code

Loading dye LD

Hyperladder 1



aspiration:

3. 3 µl of your purified PCR product


5. Carefully load your 5 µl sample of (PCR product plus loading dye) onto the



allows you to determine the size of your PCR product.


36

Section 7: Theoretical Background on Basic Molecular

Techniques

7.1 DNA Extraction

DNA Extraction or rather, nucleic extraction is the process by which nucleic acids are

liberated from and then purified away from other cellular materials. There are three basic

and followed by selective recovery of nucleic acids from the cellular lysate :

1. Physical Extraction- Breaking the cells open, commonly referred to as cell

disruption or cell lysis to expose the DNA within. This is commonly achieved by

grinding, homogenization, bead beating or sonicating the sample.

2. Chemical dissociation – the solubilisation of lipids (cellular, nuclear,

cytoplasmic and organellar membrane systems) using powerful detergent

solutions (with appropriate buffer conditions (TRIS, EDTA)) such as:

SDS (Sodium Docecyl Sulphate)

CTAB (Cetyl Trimethyl Ammonium Bromide )

For bacterial plasmid preparation:

SDS and NaOH is used to bust open the cells, followed by neutralisation and

precipitation of cell wall and protein with potassium acetate mitochondrial

DNA

3. Enzymatic dissociation

DNA associated proteins, as well as other cellular proteins, may be degraded with the

addition of a protease. Precipitation of the protein is aided by the addition of a salt

such as ammonium or sodium acetate. When the sample is vortexed with phenol-

chloroform and centrifuged the proteins will remain in the organic phase and can be

drawn off carefully. The DNA will be found at the interface between the two phases

(see below)

37

Physical Extraction: 1 – by hand

Motar and pestle

Physical Extraction: 2 – by machine

Bead Beaters Rotating incubator

38

4. Recovery of Nucleic Acids

4.1 Organic Extractions to Precipitate Proteins

phenol - (phenol + chloroform) – chloroform

Organic compounds such as phenol and chloroform are immiscible with

aqueous solutions, yet denature and precipitate out proteins from them, whilst

leaving nucleic acids untouched

Different subsets of proteins are precipitated by the different organic

compounds. Therefore mixing these compounds with a DNA lysate and then

separating the aqueous and organic phases by brief centrifugation (the

aqueous phase is the top (lighter) one) clears the aqueous phase of proteins,

which either form a pellet at the bottom of the tube or a layer at the interface

between the phases.

Because pure chloroform can be partly miscible with water, it is always used

as a 24:1 mix with isoamyl alcohol, which allows for a clearer interface

between the organic and aqueous phases. Hence, chloroform refers to this 24:1

mix.

Extraction can either be undertaken sequentially with phenol, then with a 1:1

mix of phenol-"chloroform", and finally with "chloroform", or 2 rounds of

extraction with a 1:1 mix of phenol-"chloroform" can be performed

4.2 Alcohol Precipitation

Recovery of nucleic acids by alcohol precipitation, usually ice-cold ethanol or

isopropanol will aggregate together, giving a pellet upon centrifugation. The DNA is

insoluble in the alcohol and will come out of solution, and the alcohol serves as a wash to

remove the salt previously added.

39

4.3 By selective binding

apply cell lysate to a physical matrix

DNA binds to the matrix and is separated from other components of cell

lysate on centrifugation.

wash matrix

elute nucleic acid from matrix to recover

Silica based matrix

4.4 Recovery of nucleic acids: 3 – considerations

Taxon-specfic methods (CTAB)

Environmental contamination:

other organisms

chemical contaminants eg. PCR inhibitors (humic acid in soil)

Archival DNA from collections material

Dried

Fixed

ethanol

40

formalin?

7.2 PCR – Principles and Procedures

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR

methods typically amplify DNA fragments of up to ~10 kilo base pairs(kb), although

some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. [These components

include:

DNA template that contains the DNA region (target) to be amplified.

Two primers that are complemtary to the 3’ (three prime) ends of each of the

sense and antisense strand of the DNA target.

Taq polymerase with a temperature optimum at around 70 °C.

Deoxynucleotide triphosphates (dNTPs), the building blocks from which the

DNA polymerases synthesizes a new DNA strand.

Buffer solution providing a suitable chemical environment for optimum activity

and stability of the DNA polymerase.

Divalent cations, magnesium or manganese ions ; generally Mg2+ is used, but

Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+

concentration increases the error rate during DNA synthesis[7]

Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction

tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the

reaction tubes to achieve the temperatures required at each step of the reaction (see

below). Many modern thermal cyclers make use of the Peltier effect which permits both

heating and cooling of the block holding the PCR tubes simply by reversing the electric

current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for

rapid thermal equilibration. Most thermal cyclers have heated lids to prevent

condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid

require a layer of oil on top of the reaction mixture or a ball of wax inside the tube

42

2.1 PCR-PROCEDURE

43

Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C

(or 98 °C if extremely thermostable polymerases are used), which is held for 1–9

minutes. It is only required for DNA polymerases that require heat activation by hot start

PCR.

Hot-Start PCR a technique that reduces non-specific amplification during the

initial set up stages of the PCR. It may be performed manually by heating the

reaction components to the melting temperature (e.g., 95°C) before adding the

polymerase. Specialized enzyme systems have been developed that inhibit the

polymerase's activity at ambient temperature, either by the binding of an antibody

or by the presence of covalently bound inhibitors that only dissociate after a high-

temperature activation step. Hot-start/cold-finish PCR is achieved with new

hybrid polymerases that are inactive at ambient temperature and are instantly

activated at elongation temperature.

Denaturation step: This step is the first regular cycling event and consists of heating the

reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by

disrupting the hydrogen bonds between complementary bases, yielding single strands of

DNA.

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds

allowing annealing of the primers to the single-stranded DNA template. Typically the

annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used.

Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very

closely matches the template sequence. The polymerase binds to the primer-template

hybrid and begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on the DNA polymerase

used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a

temperature of 72 °C is used with this enzyme. At this step the DNA polymerase

synthesizes a new DNA strand complementary to the DNA template strand by adding

dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-

phosphate group of the dNTPs with the 3’-hydroxyl group at the end of the nascent

(extending) DNA strand. The extension time depends both on the DNA polymerase used

44

and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its

optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

Under optimum conditions, i.e., if there are no limitations due to limiting substrates or

reagents, at each extension step, the amount of DNA target is doubled, leading to

exponential amplification of the specific DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 70–

74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-

stranded DNA is fully extended.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term

storage of the reaction.

2.2 PCR Optimization

1. PCR Primer Design Guidelines

PCR involves the following three steps: denaturation, annealing and extension. First, the

genetic material is denatured, converting the double stranded DNA molecules to single

strands. The primers are then annealed to the complementary regions of the single

stranded molecules. In the third step, they are extended by the action of the DNA

polymerase. All these steps are temperature sensitive and the common choice of

temperatures is 94oC, 60oC and 70oC respectively. Good primer design is essential for

successful reactions. The important design considerations described below are a key to

specific amplification with high yield.

Primer Length- Typical primers are 18-28 nucleotides in length. A shorter

primer such as a 15mer would have a higher chance of annealing at more than one

complementary site within the genome. This may lead to amplification of

nonspecific PCR products.

Primer Sequence

a) Avoid runs (3 or more) of C’s and G’s at the 3’ends of primers as this may

promote mispriming at G+C rich sequences.

b) A thymidine (T) at the 3' end is not recommended, since it is more prone

to mispriming than other nucleotides.

45

c) Avoid complementarity at the 3’ ends of primer pairs as this promotes the

formation of primer-dimer artefacts and reduces the yield of the desired

product. (The creation and subsequent amplification of these primer–

dimers reduces the availability of primer to the template molecule

resulting in decreased sensitivity or even failure of the PCR).

d) Avoid sequences with significant secondary structure. Primer sequences

should also be checked for self complementarity which could introduce

secondary structures like hairpin loops into the primer.

GC Content - The GC content (the number of G's and C's in the primer as a

percentage of the total bases) of primer should be 40-60 %

Primer Melting Temperature (Tm)- by definition is the temperature at which one

half of the DNA duplex will dissociate to become single stranded and indicates

the duplex stability. Primers with melting temperatures in the range of 52-58 oC

generally produce the best results. Primers with melting temperatures above 65oC

have a tendency for secondary annealing. The GC content of the sequence gives a

fair indication of the primer Tm.

Annealing temperature - The calculated Tm for a given primer pair should be

similar. For this purpose, one can use the rule of thumb calculation of 2˚C for A

or T and 4˚C for G or C (Thein and Wallace 1986). As a starting point, an

annealing temperature 5°C below the Tm can be used. This is usually the adjusted

to improve specificity and yield in a series of optimization experiments.

2. Primer Annealing

The temperature and length of time required for primer annealing depend on base

composition, length and concentration of the amplification primers. An applicable

annealing temp is 5˚C below the true Tm of the amplification primers.

Increasing the annealing temp, increases specificity. It enhances discrimination

against incorrectly annealed primers and reduces misextension of incorrect

nucleotide s at the 3’’end of primers.

Low extension temperatures together with high dNTP concentrations favours

misextension of primers and extension of misincoporated nucleotides.

46

3. Primer Extension

Extension time depends upon the length and concentration of the target sequence and

upon temperature. Primer extensions traditionally performed at 72˚C, because this

temperature was near optimal for extending primers on M13-based model template.

Length of step is dependent on your target sequence length. In general, allow 1 min. per

1,000 bp of target to be amplified. An extension time of 5-10 min. at the end of the

program allows for final extension of any unfinished products.

4. Denaturation Time and Temperature

The most likely cause for failure of a PCR is incomplete denaturaton of the target

template and/or the PCR product.

Incomplete denaturation allows the DNA to”snap back’ and, thus reduces the

product yield.

A high or too long denaturation step leads to unnecessary loss of enzyme activity.

5. Deoxynucleotide Triphosphates (dNTPs)

Deoxynucleotide concentration between 20 and 200µM each result in optimal

balance among yield, specificity and fidelity.

The four dNTPs should be used at equivalent concentrations to minimize

misincoporation errors.

Both the specificity and the fidelity of PCR are increased by using lower dNTP

concentration.

Lower dNTP concentration minimizes mispriming at non-target sites and reduces

the likelihood of extending misincoporated nucleotides.

6. Magnesium Concentration

It is beneficial to optimize the magnesium ion concentration, as this is a necessary

cofactor enzyme. A high magnesium concentration gives a higher yield, but also lower

specificity.

Magnesium ion concentration may affect all of the following:-

47

Primer annealing

Strand dissociation temperatures of both template and PCR product

Product specificity

Formation of primer –dimer artifacts

Enzyme activity and fidelity

Taq DNA polymerase requires free magnesium on top of that bound by template DNA,

primers and dNTPs. PCRS should contain 0.5 to 2.5 mM magnesium over the total dNTP

concentration. Note: the presence of EDTA or other chelators in the primer stocks or

template DNA may disturb the apparent magnesium optimum.

7. Enzyme Concentration

Recommended concentration range for Taq DNA polymerase is between 1 and

2.5 units.

Requirements may vary with respect to individual target templates or primers.

When optimizing a PCR, testing enzyme concentrations ranging from 0.5 units to

5units / 100µl and assaying the results by gel electrophoresis.

If the enzyme concentration is too high, non-specific background products may

accumulate and if too low, an insufficient amount of desired product is made.

8. Cycle Number

The optimum number of cycles will depend mainly upon the starting concentration of

target DNA when other parameters are optimized.

A common mistake is to execute too many cycles. You should not have to go over

40 cycles as a rule.

Too many cycles can increase the amount and complexity of non-specific back

ground products. Optimizing the number of PCR cycles is the best way to avoid to

amplifying background products.

Too few cycles give low product yield.

48

2.3 PCR Troubleshooting

1. The Template DNA

The amount of total DNA in a PCR has a marked effect on the outcome of a PCR

procedure.

Using too much total DNA results in packed DNA in the confined space of the

reaction vessel and can lead to false priming and even poor DNA synthesis due to

the obstructed diffusion of large Taq polymerase molecules.The concentration of

the target DNA should be balanced with the number of cycles in the reaction.

Using an elevated concentration of the target combined with the normal, or higher

than normal, number of cycles can cause the accelerated accumulation of

nonspecific products.

The accumulation of nonspecific products is often observed in a re-amplification

PCR, when the high initial concentration of the PCR fragment is accompanied by

a high number of cycles. Reducing the number of cycles may help. However, low

concentrations of primer, target, Taq, and nucleotides are recommended as these

generally ensure cleaner product and lower background.

Impurities in nucleic acid preparations or in biological samples can inhibit or

reduce the sensitivity and efficiency of PCR amplification

2. Inadequate dNTPs

An incorrect concentration of deoxynucleotide triphosphates (dNTPs) can cause

problems for the PCR procedure.

The usual dNTP concentration is between 40μM and 200μM of EACH of the four

dNTPs.

Excessive dNTP concentrations can inhibit the PCR preventing the formation of

product.

Low primer, target, Taq and dNTP concentrations are preferable as these

generally ensure cleaner product and lower background.

49

For longer PCR-fragments a higher deoxynucleotide triphosphate concentration

may be required.

A large change in the dNTP concentration may require a corresponding change in

the concentration of MgCl2.

Suboptimal concentration of nucleotides can cause incomplete primer elongation

or premature termination of DNA synthesis during the elongation step of the PCR

cycle.

3. Primer Concentration

The concentration of primer in the amplification reaction should be between 0.1 and 0.5

μM. For most PCR applications, including sensitive PCR assays and the amplification of

longer PCR products, 0.2 μM of each primer produces satisfactory results.

High primer concentrations can have the following effects:

Will promote mis-priming and accumulation of nonspecific product and may

increase the probability of generating a template-dependent artifact termed a

primer-dimer. If the primers are capable of forming dimers, raising their

concentration only results in the creation of primer-dimers and does not improve

the amplification of the desired PCR product.

Non specific products and primer–dimer artifacts generated are themselves

substrates for PCR and compete with the desired product for Taq, dNTPs and

primers, resulting in a lower yield of the desired product.

Raising the primer concentration does not therefore cause an increase in the effective

concentration of the primers. Low primer concentration generally ensures cleaner

product and lower background.

However, to amplify short PCR target sequences, careful calculation of the optimum

primer concentration is required. For example, if the target fragment length is 100bp,

a greater number of PCR product molecules is required to provide a specified amount

of amplified DNA (in nanograms) than for a larger target fragment. In order to

generate the required number of PCR product molecules, a greater number of primers

50

may be needed. Therefore, concentration of primers higher than 1μM may be necessary,

and desirable, for short target sequences.

4. Taq Concentration

In a PCR experiment approximately 1 unit of the Taq enzyme should be used for a 25μl

reaction.

Suboptimal concentration of the Taq enzyme can cause incomplete primer

elongation or premature termination of the PCR product synthesis during the

elongation step of a PCR cycle.

Too much Taq will result in an excessive background of unwanted DNA

fragments (a smear on a gel) while a huge excess may cause the reaction to fail

with no product being detected. A Taq concentration of 1 unit per 25μl reaction

ensures a cleaner product and lower background.

5. Mg Concentration

Magnesium is a required cofactor for thermostable DNA polymerases. Mg2+ in the PCR

mixture stabilizes dsDNA and raises the Tm. Mg2+ concentration therefore is an

important for controlling the specificity of the reaction.

A low Mg2+ concentration requires more stringent base pairing in the annealing

step. Too few Mg2+ ions result in a low yield of PCR product.

Too many Mg2+ ions increase the yield of non-specific products and promote

misincorporation as the fidelity of the Taq is reduced. On a gel this can appear as

a ladder or smear. The MgCl2 concentration should normally be between 1mM

and 4mM

Insufficient Mg2+ concentration in a PCR mixture can causes failure of the

reaction.

The MgCl2 concentration should normally be between 1mM and 4mM. Since

dNTPs sequester Mg2+ ions, a major change in the dNTP concentration in a

rection would require a change in the concentration of MgCl2. Similarly,

51

changing the KCl-based buffer concentration or any other component of the

PCR mix may require adjustment of the Mg2+ concentration in the reaction

mixture.

6. KCl Concentration

Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration

of 50mM. To improve the PCR amplification of DNA fragments, especially fragments in

the size range 100bp to 1000bp, a KCl concentration of between 70mM and 100mM is

sometimes recommended.

For the amplification of longer products a lower salt concentration appears to be

better.

PCR amplification of short products works better at higher salt concentrations.

This is probably because an increase in salt concentration permits shorter DNA

molecules to denature preferentially to longer DNA molecules. Shorter molecules

are therefore amplified better at higher salt concentration. It should be

remembered however that a salt concentration above 50mM can inhibit the

Taq polymerase.

An increase in KCl concentration may reduce the appearance of unwanted, long,

non-specific products.

Decreasing the KCl concentration to about 35 or 40mM, should get rid of short,

non-specific products.

In either case do not change the MgCl2 concentration. To improve the yield of a

product you can try adjusting the KCl concentration: increase it for a desired

product less than 1000bp; lower it for a desired product greater than 1000bp.

Consider the following PCR methods

Nested PCR: A second PCR is performed on the product generated from the first PCR

using primers, which are internal to the original primer pair. This improves sensitivity

without impairing specificity.

52

Semi or hemi-nested PCR: If a full nested PCR (using two internal primers) cannot be

performed, sensitivity and specificity can be improved by using just one ‘inner’ primer in

conjunction with one of the ‘outer’ primers from the first reaction.

Touchdown PCR: Begin PCR cycle with a more stringent annealing temperature and

decrease after each cycle until a predetermined annealing temperature is reached. This

allows more stringent annealing of primers to template in the first few rounds of PCR,

increasing the specificity of the PCR product. As specific products accumulate in the first

rounds, they become available as templates in succeeding rounds which have decreased

annealing temperature allowing for higher yield of product.

2.4 Frequently asked Questions (FAQ)

No Product Yield:

1. Is enzyme being added reproducibly? Consider making a master mix for multiple

PCR reactions.

2. Are proper 0.2 ml tubes being used? The 0.2 ml tubes have thinner walls and

allow for faster heat-transfer during temperature shifts.

3. Is denaturation step sufficient? Consider higher temperature or longer time.

4. Are primers stable? Reliable? Redesign or order new primers. Consider nested

PCR (2 rounds of PCR with different primer sets).

5. Are there proteases or nucleases in sample? Consider a pre-incubation at 95°C

for 5-10 min before adding Taq. Use proteinase-K and inactivate at 95°C for 5-

10 min before adding Taq.

Low Product Yield - Optimize four key parameters:

1. Annealing temperature

2. Number of PCR cycles

3. Mg2+ concentration

4. Template concentration

53

Smear of PCR products on Gel – How to get specificity:

1. Too much starting template? Check the concentration of the starting template.

Make serial dilutions of template nucleic acid from stock solutions. Perform PCR

using these serial dilutions.

2. Taq polymerase concentration too high? Use master mixes.

3. Mg2+ concentration not optimal (too high)? Use buffer that allows separate

addition of Mg2+. Perform a Mg2+ gradient from 0.5–5.0 mM (in 0.5 mM

steps).

4. Are annealing or extension steps too long? Minimize times.

5. Primer concentration not optimal or primers degraded? Repeat the PCR with

different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM

steps).

6. Too many PCR cycles? Reduce the number of cycles in steps of 3 cycles.

7. Annealing temperature too low? Optimize annealing temperature or redesign

primers. Touchdown PCR (decrease annealing temperature each cycle). Hot-start

PCR (withhold Taq until after initial 5 min. denaturation).

8. Primer design not optimal? Review your primer design, and design new primers.

9. Do primers have high complementarity to many sites on DNA? Blast primer

sequences against template DNA sequence. Consider nested primer approach.

Primer Dimer Formation

1. Are 3’ ends of primer pairs complementary? Shift primer sequence up or down

and reorder primers.

2. Are primers too short? Check primer characteristics.

3. Is template DNA concentration insufficient? Increase amount of template DNA

added or re-test template DNA concentration.

4. Are primers too concentrated? Decrease primer amount or re-dilute primers from

stock.

5. Too many cycles? Decrease number of cycles.

6. Annealing temperature too low? Increase annealing temperature or try gradient

PCR.

54

7.3 Agarose Gel Electrophoresis

Agarose electrophoresis is a method used where charged molecules in solution, mainly

nucleic acids and proteins, migrate in response to an electrical field. The rate of

migration through the electrical field depends on:

the net charge of the nucleic acid /protein

the size and shape of the molecules

the ionic strength, viscosity, and the medium through which the molecules are

moving

3.1 Factors affecting migration of nucleic acids

In an electric field, molecules migrate through the gel towards the anode

(Negative-Positive) due to their overall negative charge. Nucleic acids have a

natural negative charge due to their sugar-phosphate backbone.

Separation of molecules on the gel is influenced by the molecule size and the

effective pore size of the gel.

Increasing the agarose concentration of a gel reduces the migration speed and

enables separation of smaller DNA molecules. The higher the voltage, the faster

the DNA moves. But voltage is limited by the fact that it heats and ultimately

causes the gel to melt

Conformations of DNA plasmid that has not been cut with a restricition enzyme

will move with different speeds (slowest to fastest: nicked or open circular,

linearised or suoercoiled plasmid)

3.2. Percent agarose and resolution limits

Agarose gel electrophoresis can be used for the separation of DNA fragments ranging

from 50 base pair to several megabases (millions of bases) using specialized apparatus.

The distance between DNA bands of a given length is determined by the percent agarose

in the gel. In general lower concentrations of agarose are better for larger molecules

because they result in greater separation between bands that are close in size. The

disadvantage of higher concentrations is the long run times (sometimes days). Instead

high percentage agarose gels should be run with pulse field electrophoresis (PFE).The

55

following is a list of recommended gel percentages for the resolution of nucleic acids in

electrophoresis:

Gel percentage DNA size range bp

0.5% 1,000-30,000

0.7% 800-12,000

1.0% 500-10,000

1.2% 400 - 7,000

1.5% 200 - 3,000

2.0% 50 - 2,000

3.3 Electrophoresis Buffers

There are a number of buffers used for agarose electrophoresis. The most common being:

Tris/Acetate/EDTA (TAE). TAE has the lowest buffering capacity but provides

the best resolution for larger DNA. This means a lower voltage and more time,

but a better product.

Tris/Borate/EDTA (TBE). TBE has a greater buffering capacity and will give

sharper resolution than TAE.

3.4. Visualisation of DNA with Gel Red

Gel Red is used to make DNA or RNA bands visible in an agarose gel. It fluoresces

under UV light (excitation at 300nm, emission at 595nm) when intercalated into DNA (or

RNA)

FEATURES

Safer than EtBr (ethidium bromide)

Easy disposal - Passed environmental safety tests for direct disposal down the

drain or in regular trash

Ultra-sensitive - Much more sensitive than EtBr and SYBR Safe Sensitivity:

Bands of 0.25ng can be detected

Extremely stable -Available in water, stable at room temperature for long-term

storage and microwavable

56

Simple to use -Very simple procedures for either pre-cast and post gel staining

Perfectly compatible with a standard UV transilluminator - Gel Red replaces

EtBr with no optical setting change

Dilution of Gel Red:

1. Add 2ul of the concentrated Gel Red stock to 1000 ul of Gel loading Buffer. Mix 2ul

of this loading buffer with your sample. OR

2. Add 5 ul of Gel red to 50 ml of agarose solution.

3.5. Gel Loading Buffer

Loading dye is mixed with DNA samples for use in agarose gel electrophoresis. These

buffers serve three purposes:

They increase the density of the samples, ensuring that the DNA sinks evenly into

the well. (glycerol, sucrose or Ficoll)

They add colour to the sample, thereby simplifying the loading process.

They contain dyes that in an electric field, move toward that anode at predictable

rates.

Tracking Dyes

The most common means of monitoring the progress of electrophoretic separations by

following the migration of tracking dyes that are incorporated into the loading buffer.

Two widely used dyes displaying different electrophoretic mobilities are

bromophenol blue and xylene cyanol.

Xylene cyanol typically migrates at approximately 4kb equivalence. So do not

use this if you want fragments of 4kb.

Bromophenol blue migrates at a rate equivalent to 200-400bp DNA. If you

want to see fragments near this size (ie. Anything smaller than 600bp) then

use the other dye because bromophenol blue will obscure the visibility of the

small fragments.

57

3.6 Size Markers

There are lots of different kinds of DNA size markers. In the old days the cheapest

defined DNA was from bacteriophage so alot of markers are phage DNA cut with

restriction enzymes. Many of these are still very popular eg, lambda HindIII, lambda PstI,

PhiX174 HaeIII. These give bands with known sizes but the sizes are arbitrary. Choose a

marker with good resolution for the fragment size you expect to see in you sample lanes.

Companies have started producing ladder markers with bands at defined intervals, eg.

0.5, 1, 1.5, 2, 2.5kb and so on up to 10kb. If you know the total amount of DNA loaded

into a marker lane, and you know the sizes of all the bands, you can calculate the amount

of DNA in each band visible on the gel. This can be very useful for quantifying the

amount of DNA in your sample bands by comparison with the marker bands.

For example HyperLadder I is a typical ready-to-use molecular weight marker.

Hyper Ladder I produces a pattern of 14 regularly spaced bands, ranging from

200 to 10,000bp. The 1,000 and 10,000bp bands have the highest intensity to

allow easy identification. The size of each band is an exact multiple of 100bp.

SIZING- when using the standard loading of 5μl per lane, (720ng of DNA) each

band corresponds to a precise quantity of DNA (see figure)

58

3.7 Troubleshooting

Common problems encountered in agarose gel electrophoresis are described below, along

with several possible causes.

Poor resolution of DNA fragments. The most frequent cause of poor DNA

resolution is improper choice of agarose concentration. Low percentage agarose

gels should be used to re-solve high-molecular-weight DNA fragments and high

percentage gels for low-molecular- weight DNAs (see Table 2.5A.1). Fuzzy bands,

encountered particularly with small DNA fragments, result from diffusion of the

DNA through the gel. This is especially true when gels are run for long periods of

time at low voltages.

Band smearing. Trailing and smearing of DNA bands is most frequently observed

with high molecular-weight DNA fragments. This is often caused by overloading

the DNA sample or running gels at high voltages. DNA samples loaded into torn

sample wells will also cause extensive smearing, as the DNA will tend to run in

the interface between the agarose and the gel support.

Melting of the gel. Melting of an agarose gel during an electrophoretic separation

is a sign that either the electrophoresis buffer has been omitted in the preparation

of the gel or has become exhausted during the course of the run. For high-voltage

electrophoresis over long time periods, TBE should be used instead of TAE as it

has a greater buffering capacity. Also, minigel and midigel boxes, which typically

have small buffer reservoirs, tend to exhaust buffers more readily than larger gel

boxes.

7.4 Purification of PCR products

Template purity and concentration are the two most important factors in obtaining good

quality sequence data. Characteristics of poor quality templates:

Noisy data or peaks under peaks

No usable sequence data

Weak signal

59

4.1 Considerations when cleaning PCR products

Essential to separate the PCR product away from potentially interfering

substances that may remain in solution after the PCR reaction. Primers, dNTPS,

enzyme and salts should be removed.

Determination of the presence of a single band or multiple bands that may result

from your PCR reaction

Primers

Forward and reverse PCR primers remain in solution, will both act as sequencing

primers.

This results in multiple peaks from beginning to end as each primer can anneal to

complementary strands with different nucleotide composition (multiple reactions)

Results in overlapping fragments and unreadable data

dNTPs

Excess dNTPs will upset the specific ratios of dNTPs/ddNTPs required for

optimal extension and termination in the cycle sequencing reaction.

Salts

The processivity of the Taq polymerase used in the cycle sequencing reaction

declines in the presence of high amounts of salts

Non Specific PCR products

Primer dimer artifacts and secondary PCR products can result in poor quality

sequence data.

Nonspecific PCR products behave as templates in the sequencing reaction and

produce extension products which results in noisy data

4.2 Methods for purifying PCR products

1) Ethanol precipitation

2) Column purification

3) Gel extraction (Qiagen kit)

1) Ethanol precipitation

The PCR product is precipitated with ammonium acetate or sodium acetate and ethanol to

remove the contaminants of the PCR

Advantages

Yield products of adequate purity for direct sequencing.

60

Disadvantages

Ethanol contamination results in poor sequence data or complete failure, or

Salt contamination in DNA preps results from co-precipitation of salts in alcohol

precipitations insufficient removal of supernatant after precipitations or an

incomplete wash of the pellet with 70% ethanol

Salts can be preferentially injected and interfere with the migration of the DNA

samples

2) Column purification methods

I. Ultrafiltration

Microcon Centrifugal Filter devices - Cost £2.00 per sample

Millipore Montage PCR Clean up kit

Automated 96 well format and half volume plate format; Cost-20 p per sample

II. Affinity Column purification

Qiaquick PCR purification columns (Qiagen); Cost - £ 1.38 per sample

I. Ultrafiltration - Microcon Centrifugal filter device

Microcon Spin columns have an ultrafiltration membrane across its bottom. The

membrane has molecular size holes that allows molecules smaller than a predetermine

size to pass through it under centrifugal force. Molecules larger than the membrane cut

off size cannot pass through the membrane and are recovered from its surface. For

example YM100 (100,000 dalton nonimal molecular weight) has a nucleotide cut of for

double stranded DNA fragments of 125bp.

The process of ultrafiltration uses semi-permeable membranes and pressure to

separate molecular species on the basis of size and shape.

Microcons, are disposable units that use centrifugal force to drive such separation.

Microcon filters can be used to rapidly wash/desalt/buffer exchange, fractionate or

concentrate many aqueous biological samples including nucleic acids.

61

Advantages

Better alternative to ethanol precipitation.

Reproducible and solute recoveries typically 95% of the sample

Concentration factors as high as 100x

II. Affinity Columns – Qiagen QiAquick PCR Purification Columns

Binds SS or DS DNA from 100bp-10 Kb

Removes 99.5% of primers up to 40 nucleotides,dNTPs, salts

Recovery of DNA 90-95 %

QIAquick PCR Purification Procedure

DNA absorbs to the silca membrane in the

presence of high salt

dNTPs, primer and salts pass through the

column.

Impurities are washed away

Dry column

Pure DNA is eluted with water

62

3) Gel Extraction

This is the method of choice if your PCR reaction contains multiple bands.

Run out all of the PCR on an agarose gel

Excise out the correct size fragment and purify with Qiagen QIAquick (Gel

extraction) spin columns

Check purified DNA on a agarose gel

Check the purity and concentration of the DNA using the Nano drop

Calculate the amount of DNA required for your sequence reaction

Obviously the deciding factor on which ultrafiltation method to use could be govern by

the relative cost per sample.

Millipore PCR purification kit - 20p

Qiagen QIAquick PCR kit -£1.38

Qiagen Gel extraction kit -£1.38

Microcon filter units -£2.00

7.5 Cycle Sequencing

DNA sequencing is the process of determining the exact order of the bases A, T, C and G

in a piece of DNA. In essence, the DNA is used as a template to generate a set of

fragments that differ in length from each other by a single base. The fragments are then

separated by size, and the bases at the end are identified, recreating the original sequence

of the DNA.

Cycle sequencing is a modification of the traditional Sanger sequencing method. The

components are DNA, primer, heat resistant DNA polymerase, 4 dNTP´s, 4 ddNTP´s

(dideoxy terminator nucleotides) fluorescently labelled with four different dyes and

buffer containing MG++ and K+. The single primer binds to the complementary DNA

strand and is extended in a linear mode. This extension continues until by chance and

depending on the complementary base a particular ddNTP is incorporated. Because of the

latter’s dideoxy-configuration the polymerase cannot add any other base to this fragment

and the extension is terminated. Thus at the end of the selected number of cycles,

numerous fragments with different lengths and one labelled nucleotide at the end are

63

generated. Stoichiometric manipulation of the reaction components ensure that the

fragments of every possible length starting from n+1 say 2000 bases are generated with n

being the number of bases in the primer. The key difference between the traditional

Sanger method and cycle sequencing is the employment of a thermo stable DNA

polymerase. The advantage of using such a polymerase, is that the sequencing reaction

can be repeated over and over again in the same tube by heating the mixture to denature

the DNA and then allowing it to cool down to anneal the primers and polymerise new

strands. Therefore less template DNA is needed than for conventional sequencing

reactions. Also all 4 fluorescnce ddNTPS are in one single reaction, compared to 4

separate reactions for the old isotopic system.

After a post sequencing reaction cleanup, the samples are electro kinetically injected into

the array of 96-capillary sequencers. The negatively charged fragments migrate toward

the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be

read as the fragment’s base sequence. A laser beam excites these dye molecules as the

fragments reach a detection window, producing fluorescent signals that are collected

from all 96-capillaries at once, spectrally separated and focused onto a CCD (charge

coupled device) camera. Sophisticated optical and electronic devices produce a colour

readout that is translated with the help of sequence analysis software into a sequence as

we see it.

• template requirement (2-3ng / 100 bp of PCR product)• secondary structure

Cycle sequencing reaction

64

TCGA

Equivalence of manual and automated sequencing

65

O

|H

O O O H| | | |

O = P - P - P - O - C - H| | |O O O

|O|H

base

Deoxy-ribonucleotideRIBONUCLEOTIDE

O

|O|H

O O O H| | | |

O = P - P - P - O - C - H| | |O O O

|O|H

base

Chain grows in thisdirection

|O H| |

O = P - O - C - H|O(-)

O H| |

O =P - O -C - H|O(-)

O

base

|H

|O|H

5’

3’

phosphodiesterlink

O

base

|H

|

nextdNTPadded here

DNA replication

O

|H

O O O H| | | |

O = P - P - P - O - C - H| | |O O O

|H

base

Di-deoxy-ribonucleotide

O H| |

O = P - O - C - H|O(-)

O

base

|H

|O|H

|

O O O H| | | |

O = P - P - P - O - C - H| | |O O O

O

base

|H

|H

O O O H| | | |

O = P - P - P - O - C - H| | |O O O

O

base

|H

|H

phosphodiester link

chain grows

no phosphodiester link

chain is terminated

66

5.1 Preparing the Samples for Cycle Sequencing 2-2 Applied Biosystems 3730/3730xl

DNA Analyzers Sequencing Chemistry Guide

Template Types

The following templates may be used with the BigDye® terminator chemistry:

• PCR product

• Single-stranded DNA (e.g., M13)

• Double-stranded DNA

• Large DNA (e.g., BACs, PACs, YACs, cosmids, and fosmids)

• Bacterial genomic DNA

Template Quality

The quality of DNA in a reaction can affect the performance of the DNA Analyzer.

When preparing DNA templates, it is critical to avoid the following:

• Residual salts

• Proteins

• Residual detergents

• Residual RNA

The presence of residual salts, proteins, RNA, and detergents can interfere with

capillary electrophoresis and electrokinetic injection. Your current template

purification methods may have to be modified to remove residual salts, proteins, and

detergents. The following are characteristics of poor quality templates:

• Noisy data or peaks under peaks

• No usable sequence data

• Weak signal

Effect of Residual Salts

Capillary electrophoresis is especially susceptible to salt in samples, either from

template preparation, from cycle sequencing reactions, or from precipitation methods

using salts. The negative ions in salts can be preferentially injected into the capillary

array during electrokinetic injection, leading to lower signal. In addition, the negative

ions compete and interfere with the injection of larger DNA extension fragments,

leading to shortened read lengths.

67

Effect of Proteins

Many DNA preparation methods for sequencing require the recovery of DNA from

lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the

DNA samples. Protein can be injected and adhere to the walls of the capillary array,

adversely affecting data resolution and capillary array lifetime.

Effect of Residual Detergents

Some methods of template preparation, such as the Thermomax method for M13

preparation, use detergents such as Triton X-100 to lyse the protein coat of phage

particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in

plasmid purification protocols to lyse bacterial cells. Small, negatively charged

detergents may be preferentially injected over DNA during electrokinetic injection. If

present at high levels, detergents such as Triton X-100 and SDS will adversely affect

the life of the capillary array and the quality of the sequencing data.

Effect of Residual RNA

Residual RNA that is present in DNA template preparations competes with the DNA

for injection into the capillary array. Residual RNA has the same effect as excess salt,

that is, decreased signal and shortened read lengths.

Template Quantity

Effect of Too Little Template

Too little template or primer in cycle sequencing reactions reduces the signal strength

and therefore the peak height of reaction products. In the worst case, the signal-to noise

level decreases so that bases cannot be called.

Effect of Excess Template

Excess template can affect data quality when:

• present in sample loaded onto the DNA Analyzer

• used in excess in the cycle sequencing reaction

Excess template inhibits the injection of labeled extension fragments, thus affecting

signals generated from this instrument. Excess template can behave similarly to

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proteins and accumulate in the capillary array, which adversely affects data

resolution and capillary array lifetime.

Excess template used in the cycle sequencing reaction results in generation of short

extension fragments. During electrokinetic injection, short fragments are injected

more efficiently resulting in “top heavy” peak characteristics and shortened reads.

(This phenomena becomes more pronounced with increased dilution of BigDye

Terminator.

Raw data from a BigDye terminator reaction containing excess DNA Template

Below, analyzed data from a BigDye Terminator reaction contains excess template (this is from

the same sample shown in above). The peaks are clearly off-scale and have overall shortened read

lengths. The presence of excess template in the reaction and the preferential electrokinetic

injection of small DNA fragments cause this effect

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

69

QuickTime™ and aTIFF (Uncompressed) decompressor


70

7.6 Sequencher DNA sequence assembly and analysis software

1. Sequence Editing

Sequencher gives you the DNA sequence editing tools you need to know that a sequence

is absolutely correct. You can look at your chromatogram data one sequence at a time or

view multiple aligned chromatograms in both forward and reverse orientations

2. Sequence Trimming

Automated DNA sequencers occasionally produce poor quality reads, particularly near

the sequencing primer site, and toward the end of longer sequence runs. The sequences of

clones from DNA libraries frequently contain vector sequence, polyA tails, or other

unrelated sequence. Introns and primer sequence frequently flank the sequence of

QuickTime™ and adecompressor


71

amplified exons. Unless removed by trimming, any of these artifacts will distort your

sequence assembly and downstream sequence analysis.

Sequencher provides simple-to-use but powerful tools that help you trim poor quality or

ambiguous data:

Trim Ends removes misleading data from the ends of sequencing fragments.

Trim Vector removes sequence-specific data contaminating the ends of your sequences.

Trim to Reference eliminates the ends of sequences that extend beyond an assembled

reference sequence.

Sequence Assembly

Sequencher's intuitive controls allow you to set your

sequence assembly parameters and adjust them within

seconds, allowing you to assemble your DNA

fragments quickly and accurately. Sequencher will





72

automatically compare the forward and the reverse-complement orientations to assemble

the best possible contigs, so you can assemble DNA sequences regardless of orientation.

Apply Sequencher's versatile assembly tools to:

Compare gene variants to a reference sequence

Confirm vector constructs

Assemble viral and bacterial genomes

Cluster tens of thousands of sequences from cDNA libraries

Assemble cDNA to genomic sequence

Create a primer map

4. Support for Confidence Values

Sequencher displays confidence and summary confidence information (if available

in your DNA sequence files) in the Project window, the Sequence Editor, and the

Sequence Get Info window, so you can easily monitor the quality of your data.



73

Genebank Feature Handling

Sequencher imports GenBank Features in your files (if present) and has a variety of tools

to help you manage, add, or edit Features. You can import Genbank Feature keys, or

designate a key for your own features. The Define Feature Key Default Style user

preference lets you set up your own colors and Feature annotations, so you could

distinguish introns from exons, and variants from sequencing errors for example.





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7.7 The Basic Local Alignment Search Tool (BLAST)

The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity

between sequences. The program compares nucleotide or protein sequences to sequence

databases and calculates the statistical significance of matches. BLAST can be used to

infer functional and evolutionary relationships between sequences as well as help identify

members of gene families. There are many different variations of BLAST available to use

for different sequence comparisons, e.g., a DNA query to a DNA database, a protein

query to a protein database, and a DNA query, translated in all six reading frames, to a

protein sequence database. For a detail explanation of BLAST, please refer to the NCBI

Handbook:

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch16

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APPENDIX 1- Buffers

1. Moles and Molar solutions (unit = M = moles/L)

Sometimes it may be more efficient to use molarity when calculating concentrations. A

mole is defined as one gram molecular weight of an element or compound, and

comprised of exactly 6.023 x 10^23 atoms or molecules (this is called Avagadro's

number). The mole is therefore a unit expressing the amount of a chemical. The mass (g)

of one mole of an element is called its molecular weight (MW).

The number of moles in an arbitrary mass of a dry reagent can be calculated as:

# of moles = weight (g)/ molecular weight (g)

Molarity is the unit used to describe the number of moles of a chemical or compounds in

one liter (L) of solution and is thus a unit of concentration. By this definition, a 1.0 Molar

(1.0 M) solution is equivalent to one formula weight (FW = g/mole) of a compound

dissolved in 1 liter (1.0 L) of solvent (usually water).

Example 1: To prepare a liter of a simple molar solution from a dry reagent

Multiply the formula weight (or MW) by the desired molarity to determine how many

grams of reagent to use:

Chemical FW = 194.3 g/mole; to make 0.15 M solution use

194.3 g/mole * 0.15 moles/L = 29.145 g/L

Example 2: To prepare a specific volume of a specific molar solution from a dry

reagent

A chemical has a FW of 180 g/mole and you need 25 ml (0.025 L) of 0.15 M (M =

moles/L) solution. How many grams of the chemical must be dissolved in 25 ml water to

make this solution?

76

#grams/desired volume (L) = desired molarity (mole/L) * FW (g/mole)

by algrebraic rearrangement,

#grams = desired volume (L) * desired molarity (mole/L) * FW (g/mole)

#grams = 0.025 L * 0.15 mole/L * 180 g/mole

after cancelling the units,

#grams = 0.675 g

So, you need 0.675 g/25 ml

2. Buffers

1. 50 x TAE (Tris-Acetate EDTA) Buffer pH 8.0 ( 1 litre)

242 g Tris base in 750 m ddH2O

57.1 ml Glacial acetic acid

100 ml 0.5M EDTA pH 8.0

Stir, do not adjust the pH

Make up to 1000 ml

Sterlize by autoclaving and store at room temperature.

1 x TAE

Dilute to 1x concentration for the use in electrophoresis by taking 20ml of 50x buffer and

making the volume to 1000ml by adding 980 ml of ddH2O.

Final solute concentrations are 40 mM Tris acetate and 1 mM EDTA. The buffer is now

ready for use in running an agarose gel.

2. 0.5M EDTA, pH8

Dissolve 186.1g EDTA (Ethylenediaminetetra-acetic acid disodium salt) in 800ml

distilled water. Stir, on a magnetic stirrer with a 'flea' (magnetic stirring bar), and with the

pH electrode in the buffer gradually add solid sodium hydroxide pellets (~20g of NaOH

77

pellets). Measure the pH and adjust until it is stable at pH8 with 5M NaOH solution. (As

you add the NaOH the pH will come down. This allows more of the EDTA to dissolve

and the pH will rise again - so you have to continue until the solution is clear and the pH

is stable) Adjust volume to 1 liter with distilled water. Sterlize by autoclaving and store

at room temperature.

3. TE (10mM Tris-HCL pH 7.5 / 0.1 mM EDTA pH 8.0)

Stock Buffers required: 1 M Tris-HCL pH 7.5 and 0.5 M EDTA pH 8.0

For 50ml of TE buffer:

VOLUME REQUIRED FOR 50 ML

1M Tris-HCL pH 7.5 O.5 ml (500 µl)

0.5 M EDTA pH 8.0 0.01 ml (10 µl)

Sterile distilled water 49.49 ml

4. 2 x CTAB Extraction Buffer

Weigh out chemicals with a face mask to avoid inhalation of dust.

Tris (hydroxymethyl) amino methane 2.42 g

Sodium Chloride 16.42 g

Ethylenediaminetetra-acetic acid, disodium salt 1.2 g

Hexadecyltrimethyl-ammonium bromide (CTAB) 4.0 g

2-ß Mercaptoethanol 0.4 ml

For 200 mls of 2xCTAB: First dissolve the TRIS in 150 ml distilled water, then add

EDTA, then the remaining chemicals and make up to 200 ml mark, using distilled water.

Heating the solution gently may help to make the CTAB dissolve more quickly.

5. Phenol/ Chloroform / isoamyl alcohol (25:24:1) –This can be bought commercially

Prepare Phenol /chloroform/ isoamyl alcohol in 24: 1 ratio.

For 50 ml mix the following: Phenol 25 ml

Chloroform 24 ml

Isoamylalcohol 1 ml

Mix and store at 4°C in a dark bottle.

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6. Chloroform / isoamyl alcohol (24:1) –This can be bought commercially

Prepare chloroform/ isoamyl alcohol in 24: 1 ratio.

For 50 ml mix the following:

Chloroform 48 ml

Isoamylalcohol 2 ml

Mix and store at 4°C in a dark bottle.

General Note: The Molecular Cloning: A Laboratory Manual (Third Edition)

(Joseph Sambrook, Peter MacCallum, David Russell) Volume 3 contain the recipes

and methodology to prepare standard buffers used in molecular biology.

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Appendix 2: Tissue and DNA Storage

I. Tissue Storage

1. Cryo-preservation

The ideal storage for molecular specimens is by using cryo-preservation, storage in liquidnitrogen or at -80ºC.

2. Storage in ethanol

Ethanol preserved samples are regularly used in DNA molecular studies. However thequality of the alcohol used can play a large part in the condition of the DNA in aspecimen. Preservation in absolute ethanol offers the best means of preserving both theDNA and the gross morphology of a specimen. DNA extracted from specimens preservein absolute ethanol are of:

High molecular weight High quality

Disadvantages of ethanol preservation:

Can cause extensive tissue shrinkage Colour loss Extract cellular components such as lipids

3. Storage in RNA later

This reagent was designed for the storage of tissue samples from which you can extractRNA. However tissue stored in this buffer can be also used to extract DNA successfullyof high molecular weight and quality

RNA later ® is an aqueous, non toxic, tissue storage reagent that rapidly permeates mosttissues to stabilize and protect RNA in fresh specimens. RNA later eliminates the need toimmediately process or freeze samples; the specimen can simply be sub- merged in RNAlater and stored for analysis at a later date.

Storage and stability Store RNA later at room temperature. It is guaranteed for 6 months from the date of

receipt, properly stored. Samples in RNA later can be stored for extended periods under conditions where

RNA degradation would normally take place rapidly. Tissues can be storedindefinitely in RNA later at –20°C or below.

RNAlater can be safely discarded down the sink and flushed with water.

Materials compatible with RNA later

RNA later can be used for RNA preservation with most tissues, cultured cells, bacteria,

80

and yeast. RNA later may not be effective in tissues that are poorly penetrated by thesolution, such as waxy plant tissue and bone. RNA later has been extensively tested with

animal tissues including, brain, heart, kidney, spleen, liver, testis, skeletal muscle, fat,lung, and thymus. RNA later has also been proven effective for RNA preservation in E.coli, Drosophila, tissue culture cells, white blood cells, and some plant tissues.

4. Whatman FTA Cards

FTA cards are designs for room temperature collection, shipment, archiving andpurification of nucleic acids from a wide variety of biological samples for PCR analysis:

Blood

Cultured cells

Buccal cells

Plant material

Bacteria

Plasmids

Microorganisms

Solid tissue

Viral particles

FTA cards are impregnated with a patented solution that lyses cell membranes anddenature proteins upon contact. Nucleic acids are immobilized and protected from UVdamage and microbial fungal attack. FTA cards are available in several sizes to meetspecific needs. Since captured nucleic acids are stabilized, FTA Cards facilitate samplecollection in remote locations and simplify sample transport. For example, you cancollect samples in the field without worrying about immediate refrigeration. Ship yoursamples back to the laboratory without expensive special handling or dry ice and processat your convenience.

Store nucleic acids at room temperature for years

Genomic DNA stored on FTA Cards at room temperature for over 17 years (and counting) has been successfully amplified by PCR. RNA, being chemically less stablethan DNA, is best analyzed upon return of samples to the laboratory. Frozen storage ishelpful for RNA preservation.

Sample integrity is optimized when FTA Cards are stored in a Multi-Barrier Pouch with aDesiccant Packet.

FTA Cards offer a compact room-temperature storage system that reduces the need forprecious freezer space.

II. DNA Storage

81

Extracted DNA should be stored as follows:

-20ºC or below for long term storage

DNA for storage should be re-suspended in a buffer solution such as 10 mM TrisHCL pH 7.5 rather than water. If the pH of water is not neutral but slightly acidicthis can cause the DNA to degrade upon storage.

DNA can be stored lyophilized at -20ºC or below for long term storage

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Appendix 3: IUB Codes -Nomenclature for Incompletely SpecifiedBases in Nucleic Acid Sequences

IUB Base Code Guide

Standard Bases

Mixed Bases (Wobble)

Code BaseB C, G or TD A, G or TH A, C or TV A, C or GR A or GY C or TK G or TM A or CS G or CW A or TN Any base

A, C, G or T

Code BaseA AdenosineC CytidineG GuanosineT Thymidine

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Appendix 3: Consumables and Laboratory equipment

I-Consumables

Description Supplier pack size

KITSQIA quick PCR purification kit Qiagen 50QIA quick PCR purification kit Qiagen 250QIA quick gel extraction kit Qiagen 50QIA amp DNA mini kit Qiagen 50QIA amp DNA mini kit Qiagen 250DNeasy blood and tissue kit Qiagen 50DNeasy blood and tissue kit Qiagen 250Taq DNA polymerase Qiagen 1000UDNEasy Plant mini kit Qiagen 50

Oligonucleotides

oligo's unmodified ANY

TaqGoTaq Flexi DNA polymerase Promega 100u - each

dNTPdNTPs set 100 mM Bioline 4x25umol (4x250ul)

Enzymes

Proteinase K Sigma Genosys 500mg

Gel Markers

Gel marker - Hyperladder IV Bioline 200 LanesGel marker - Hyperladder I Bioline 200 Lanes

GelRed gel stain CambridgeBioscience

100ul

Tips

0.1-10ul refill tips Starlab 10x961-200ul refill tips Starlab 10x96200-1000ul refill tips (graduated) Starlab 10x960.1-10ul filter tips racked Starlab 10x96

0.5-10ul filter tips racked Starlab 10x961-20ul filter tips racked Starlab 10x9620-200ul filter tips racked Starlab 10x96200-1000ul filter tips racked Starlab 10x96Fine tip mini pastettes Fisher 500 pack

Tubes0.2ml thin wall 8 tubes Strip Appleton Woods 120Flat cap thin wall 8 cap strip Appleton Woods 120

0.2ml PCR Tubes, Flat Cap Fisher 10000.5ml microcentrifuge tubes Flat cap Starlab 5x2001.5ml microcentrifuge tubes flat cap Starlab 500 pack2ml microcentrifuge tubes flat cap Fisher 1000 pack15ml centrifuge tubes -Falcon fisher 10 rack of 5050ml centrifuge tube, non-skirted fisher 500 pack7ml Aseptic, polypropylene, Bijou containers Fisher 1800 pack30ml Disposable, Polystyrene, sterile universals Fisher 400 packGlovesnitrile gloves small Semperguard Fisher 1000 pack

nitrile gloves medium Semperguard Fisher 1000 pack

84

Description Supplier pack sizenitrile gloves large Semperguard Fisher 1000 packnitrile gloves xlarge Semperguard Fisher 1000 pack

Miscellaneous

Super Glossy Sony Thermal Paper UPP-110HA UVP 5 rolls in a boxSony Thermal Printer paper Sony UPP-110HA UVP 5 rolls in a boxweighing boats anti static 30ml VWR 500 pack

(80x60x14mm)weighing boats anti static 100ml Fisher 1x 250 packdust masks (budget)autoclave tape - Steam ANYparafilm 100mm wide 38m long VWR 1 Roll (55mx19mm)saranwrap fisher 1aluminium foil 75M x 450mm Western Laboratory 36 boxesAutoclave bag, polypropylene, without text,

Chemicals VWR 500 pack(600x780mm)

Agarose VWR 350 pack(700x1100mm)

Ethanol absolute

Disposable Pellet Pestles- Hand-operated or motor-driven grinders for resuspending

pellets or disrupting soft tissue in microcentrifuge tubes. Pestle ends are specially

designed to mate with 0.5 mL or 1.5 mL microtubes. Shaft is convenient length and

diameter for gentle manual back-and-forth rotation or more vigorous motorized

operation.



85

II –Laboratory equipment

1. Cold storage Freezers & refrigerators

1. Ultracold -80 Freezer with a racking system

2. -20°C freezer with a racking system

3. Refrigerator +4ºC

2. Autoclave

3. Ice Maker Machine

Large volume refrigerated centrifuge-high speed

4. Laminar flow hood

5. Fume hood

6. Drying cabinet

7. Dish washer

8. Microwave

9. Magnetic stirrers with heating

10. Vortex mixer

11. Balances

Fine balance (5 dp)

Balance (3dp) or



86

New Top of the range balance has dual mode from 5dp tp 3 dp depending on

weight and anti static. Model: Qubis Sartorius Genuis balance MEM4145-

supplied via VWR

12. Centrifuges:

Microcentrifuges

Bench centrifuges

Refrigerated Benchtop centrifuge

High Speed Centrifuge

13. Experion Bioanalyser-measures DNA/RNA/Proteins

14. Thermomixer Comfort- Supplier Eppendorf







87

16. Nanodrop-spectrophotometer-Supplier Thermo Scientific

ND1000-single channel

ND8000-8 channel

17. PCR machines-Best to get gradient machines this is useful when optimising the

annealing temperature for new primers. For example:

Applied Biosystems Verity 96

Gstorm- GS00001

GS482 twin block

GS482 twin block allows you to run to

separate PCRs independently

18. Speedvac (vacuum concentrators)- to evaporate alcohol or dry down large volumes

19. Precellys 24-tissue homogeniser-Supplier Bertin Technologies







88

20. UV transilluminator –short wave

21. Long wave UV reader

21. UVP Gel documentation / digital camera system

22. Water baths

23. Dri heating block

24. Incubators

Standard

Air incubator

Cool incubator

Shaking incubator

25. pH meter

26. PCR cabinet- use to set up PCRs to reduce contamination





89

27. Distilled water system

28. Horizontal electrophoresis unit with various comb sizes

Mini

Midi

Maxi

29. Mini variable power supply for electrophoresis gel tanks

30. Adjustable Variable pipettes

1-10 µl

2-20 µl

10-100 µl

50-200 µl

100-1000 µl

31. Freezer Storage boxes: Supplier Star lab

(1) 1.5 and 2 ml tubes (PP)

Catlog no: I2381-5040 StarStore 81-Place Storage Box

(2) Storage box no dividers for 15 ml & 50 ml Falcon tubes

Catlog no: E2300-5005

1. 2.







90

32. Tube Racks

Racks for microcentrifuge tubes – 0.2 ml, 0.5 ml, 1.5 ml, 2ml

StarRack™ 96

96-place double-sided rack

Transparent lift-off lid

Holds 96 x 0.5ml tubes on one sideor 96 x 1.5/2.0ml tubes on the other

Optional, 10-place acrylic rack available to help keep your racks tidy

Temperature range: -90°C to 121°C

Dimension (WxDxH): 222 x 114 x 28mm (without lid) / 50mm (with lid)

PCR tube rack-0.2 ml

Racks for 15 ml & 50 ml tubes

33. Ice Buckets











91

34. Lab Pal Label PrinterThe LAB PAL label printer is portable, easy-to-use and makes identifying your samplesmore quick and efficient. The long-lasting labels are designed to withstand extremelaboratory environments, such as liquid nitrogen, hot water baths, freezers andautoclaves. They also resist most chemicals, including Xylene, Alcohol, Ethanol andDMSO.

35. Howie Lab coats with elasticated cuffs.

36. Safety eye protection-goggles

37. Spatulas

38. Magnetic Stir bars

39. Robotic Station- Supplier Hamilton Robotics

DNA extractions

PCR set up

PCR clean up etc.



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