pre-lab e 5: restriction enzyme digest and plasmid mapping ... · biol 2281, spring 2016 e 5:...

Pre-Lab E 5: Restriction Enzyme Digest and Plasmid mapping (10 pts) Name: _______________________________; Lab Section: _______________; Grade: _______

1. Describe the function of DNA ladder (1 pts).

2. Describe the function of loading dye (1 pt). 3. DNA fragments are __________ charged, they will be drawn toward the ________ electrode

(1 pt). 4. Which one is not the essential component of a restriction digest reaction? (1 pt)

a) DNA plasmid b) Appropriate buffer (10X) c) Loading dye d) Restriction Enzyme

5. What is the correct amount of agarose to make 55 ml of 0.75 % agarose gel? (show

calculation and unit, 2 pt.)

6. The plasmid size of pGLO is 5371 bp and the restriction sites for for PstI are at 2106 and 3181. Calculate to predict the sizes of fragments when pGLO is digested with PstI. (1pt)

7. Short Answer: How is DNA visualized on agarose gel? (include reagents and equipments in

your answer) (2 pts) 8. True or False: (1 pt, correct the errors to obtain full credits)

________ . In general, restriction sites are palindromic, meaning the sequence of bases reads the same forwards as it does backwards on the same DNA strand.

Biol 2281, Spring 2016 E 5: Restriction Enzyme Digest and Plasmid Mapping

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Experiment 5: Restriction Enzyme Digest and Plasmid Mapping

Objectives: By the end of this lab, you will be able to: Understand the use of restriction enzymes as biotechnology tools Become familiar with the principles and techniques of agarose gel electrophoresis Estimate DNA fragments sizes from agarose gel data Use a restriction map to predict how many fragments will be produced in a given restriction

digest. Note: The introduction was adapted from Restriction Digest and Analysis of Lambda DNA Kit, Bio-Rad

Laboratories, Inc. Duplication of any part of the document is permitted for classroom use only. Introduction This excercise introduces you to some important principles of genetic engineering. Specifically, the functions of restriction enzymes and their use as molecular biology tools will be stressed. Using agarose gel electrophoresis, you will examine the digestion patterns and determine the sizes of unknown DNA fragments. Restriction enzymes were a catalyst for the molecular biology revolution, and now hundreds of such enzymes are commercially available. In this investigation, the restriction enzymes EcoRV, and Pst I will be used to digest a plasmid, a small circular piece of DNA. Gel electrophoresis will be employed to separate the resulting DNA fragments, and ethidium bromide will be used to stain the DNA fragments for visualization. Restriction Enzymes The ability to cut and paste, or cleave and ligate, a functional piece of DNA predictably and precisely is what enables biotechnologists to recombine DNA molecules. This is termed recombinant DNA technology. The first step in DNA splicing is to locate a specific gene of interest on a chromosome. A restriction enzyme is then used to cut out the targeted gene from the rest of the chromosome. This same enzyme is also used to cut the DNA of the recipient into which the fragment will be inserted. Restriction enzymes are proteins that cut DNA at specific sites. Restriction enzymes, also known as restriction endonucleases, recognize specific sequences of DNA base pairs and cut, or chemically separate, DNA at that specific arrangement of base pairs. They were first identified in and isolated from bacteria that use them as a natural defense mechanism to cut up the invading DNA of bacteriophages — viruses that infect bacteria. Any foreign DNA encountering a restriction enzyme will be digested, or cut into many fragments, and rendered ineffective. These enzymes in bacteria make up the first biological immune system. Each restriction enzyme is named after the bacterium from which it is isolated. For example: EcoRI = The first restriction enzyme isolated from Escherichia coli bacteria EcoRV = The fifth restriction enzyme isolated from Escherichia coli bacteria

PstI = The first restriction enzyme isolated from Providencia stuartii bacteria Each restriction enzyme recognizes a specific nucleotide sequence in the DNA, called a restriction site, and cuts the DNA molecule at only that specific sequence. Many restriction enzymes leave a short length of unpaired bases, called a “sticky” end or “cohesive” end, at the DNA site where they cut, whereas other restriction enzymes make a cut across both strands creating double-stranded DNA


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fragments with “blunt” ends. In general, restriction sites are palindromic, meaning the sequence of bases reads the same forwards as it does backwards on the opposite DNA strand. For example, here is a list of enzymes and the sites where they cut: EcoRI 5’G A-A-T-T-C 3’ EcoRV 5’G-A-T A-T-C 3’ PstI 5’C-T-G-C-A G 3’ 3’ C-T-T-A-A G 5’ 3’ C-T-A T-A-G 5’ 3’G A-C-G-T-C5’ Setting up a simple restriction digest requires four mandatory ingredients:

• DNA: DNA that is free from contaminants such as phenol or ethanol. Excessive salt will also interfere with digestion by many enzymes.

• An appropriate buffer: different enzymes cut optimally in different buffer systems, due to differing preferences for ionic strength and the type of cations.

• The restriction enzyme • Deionized water: Specific amount of water is required to make up the final volume for a digest

reaction. In this investigation, students observe the effects of two restriction enzymes on pGLO plasmid DNA. pGLO plasmid DNA is 5,371 base pairs, each restriction enzyme will cut the DNA one or several times and generate restriction fragments of different sizes. In this activity, three separate samples of plasmid DNA will be cut using two different restriction enzymes and the combination of them. Each sample produces DNA fragments whose sizes can be estimated on an agarose gel electrophoresis. Electrophoretic Analysis of Restriction Fragments If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments of DNA. Therefore, if a given piece of linear DNA is cut with a restriction enzyme whose specific recognition sequence is found at five different locations on the DNA molecule, the result will be six fragments of different lengths. The length of each fragment will depend upon the location of restriction sites on the DNA molecule. A DNA fragment that has been cut with restriction enzymes can be separated using a process known as agarose gel electrophoresis. The term electrophoresis means to carry with electricity. Agarose gel electrophoresis separates DNA fragments mainly by size. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole (anode) when placed in an electric field. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely proportional to the log of the molecular weight (size or length). Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single bands of DNA. A 500 bp ladder containing 16 bands in 500bp increments (the smallest one is 500 bp) will be used to estimate the sizes of DNA fragments on a 0.8% - 1.0% agarose gel. Gels of 0.8-1.0% (w/v, 1 gram of agarose in 100 ml running buffer) agarose will separate fragment sizes ranging from around 500 base pairs (bp) to around 10,000 bp (or 10 kilobases, kb). Large fragments (e.g., >10 kb) will be better separated in lower percentage agarose gel, such as a 0.5% gel.


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On the other hand, separation of very small (less than 1 kb) can be better achieved in higher percentage agarose gel, such as a 1.5-2% gel. Making DNA Visible DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. The loading dye (or loading buffer) does not stain the DNA itself but makes it easier to load the gels and monitor the progress of the DNA electrophoresis. Loading buffer usually contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells. It also contains one or two tracking dyes, which migrate in the gel and allow visual monitoring of how far the electrophoresis has proceeded. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. The “faster” dye, Bromophenol blue, comigrates with DNA fragments of approximately 300 bp, while the “slower” dye, Xylene cyanol, comigrates with DNA fragments approximately 9 kb in size. Visualization of the DNA sample requires the use of a transilluminator equipped with a UV lamp. A mid-wavelength (260-369 nm) UV lamp emits light in the optimum range for viewing ethidium bromide-stained gels. You will view the gels while wearing safety glasses. This fluorescent dye intercalates between bases of DNA and RNA It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide (EB). You must wear disposable gloves during the lab because EB is a mutagen and a suspected carcinogen. You can compare the DNA restriction patterns of the different samples of DNA when the bands are visible using UV lamp. In addition to electrophoresis, most of the procedures in this exercise employ microchemical techniques; i.e., using very small amounts of reagents such as DNA and enzymes. In research laboratory, many reactions of restriction digest require the addition of only one microliter (1 µl, one one-millionth of a liter) of some components, so it is essential that you become familiar with measuring small volumes accurately and reproducibly. Procedures

Materials and Equipments

1) each work station ( for 2 students) a. 4x loading dye (next to water bath) b. A yellow box of pipet tips c. micropipetter: p20 d. a microcentrifuge tube rack e. marker f. Three microcentrifuge tubes g. A tube containing 30 µl of pGLO plasmid (0.03 µg/µl) h. One white container for used micropipette tips i. diluted EcoRV and PstI restriction enzymes in ice buckets at sink area

2) every group (4 students will share one gel)

a. Agarose (next to the balance) b. 125 ml flask for melting agarose c. 50 ml cylinder (near the sink) d. 400 ml of 0.5XTBE buffer (near the sink) e. Gel apparatus f. Power supply g. 10-teeth comb


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Note: the gel apparatus comes as a set, i.e. the lid matches the tank. Clean the gel apparatus and slide the lid back onto the tank to prevent mismatches!

1. Label three microcentrifuge tubes as follows:

P (PstI digest) E (EcoRV digest) D (double digest of PstI and EcoRV, two enzymes pre-mixed)

a) Transfer 10µl of pGLO plasmid to each tube. (check the volume setting on micropipette)

b) Restriction enzymes have been pre-mixed with buffer by instructor and placed in ice buckets near front

sink. Using a red micropipette and a fresh tip for each tube, add 5 µl of diluted restriction enzymes according to the list above.

c) Flick the tubes to mix well. Spin the tubes for 5 seconds in the microcentrifuge. The tubes should be placed in a balanced configuration. Do not open the lid if the motor is still running.

d) Incubate the tubes at 37ºC water bath for 50 minutes.

* 1 unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete digest on 1µg of substrate DNA in 60 minutes at the appropriate assay temperature in a 50µl reaction volume.

2. [one gel per 4 students] Assemble the gel apparatus using a ten slot comb.

a) Place gel casting tray on the top of gel support deck, the open ends of the tray should be placed next to the sides of the chamber. Insert the comb into the end slot.

b) Wear disposable gloves to prepare 50 ml of the melted 0.8% ( w/v, 0.8 gram/ 100 ml buffer) agarose solution . The solution contains _____ g of agarose and 50 ml of 0.5X TBE buffer (4.5mM Tris, 4.5mM boric acid, 0.1 mM EDTA, pH 8.3). Boil the buffer and dissolve agarose completely using microwave with 30 seconds intervals, the suspension needs to be completely clear.

c) Ask TA to add 1µl ethidium bromide to your melted gel solution. Pour the gel solution to the casting tray. Rinse small flask with water.

d) Demonstrations:

e) After the solidification has occurred, lift the casting tray and rotate it 90 degree. Rinse the comb with water. Under most conditions, DNA has a negative charge caused by the phosphate groups on the outer surface of the molecule. Negatively charged DNA molecules will migrate toward the positive electrode during electrophoresis. You should place the end of gel with wells near _________(cathode [black] or anode [red]).

f) Slowly fill the electrophoresis chamber with the 0.5X TBE buffer until the gel is completely submerged. The buffer should be about 3-4 mm above the gel surface.

3. Retrieve your thee digested samples from the 37ºC water bath. Obtain one tube of 4X TBE loading dye (0.04% bromophenol blue, 20% glycerol, 0.04% xylene cyanol) for two students .


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4. (Your bench) Using a fresh tip for each digested sample, add 5 µl of 4X TBE loading dye. Flick the tubes to mix well and spin for 5 second in the microfuge.

5. Set micropipette (p20) at 20 µl, load your three samples with fresh tips in the appropriate lanes following gel diagram Fig.1 as a guide. Do not leave space between each sample.

6. Ask TA to load 5 µl of the 500bp DNA ladder (Bio-rad) and uncut pGLO plasmid in the middle lanes. The DNA ladder contains 16 bands in 500bp increments; the smallest one is 500 bp.

7. When the samples have been loaded, set the power supply to 130V. Run the bromophenol blue dye to within 2 cm of the positive electrode end of the gel (35 minutes approximately). Predict the sizes of fragments in base pairs based on the information in Fig 3.

8. Visualize the DNA bands on a UV light box. Wear proper eye protection. You may trace the band pattern on a piece of plastic wrap overlaying the gel or take a picture using a digital camera. Note: The migration of DNA molecules in agarose gels is roughly proportional to the inverse of the log of their molecular weight (corresponding to the size of the fragments). The size of the DNA fragments can be determined by comparing the migration distances (mm) to the standard curve (log bp vs. migration distance) of the marker if the electrophoresis is run longer to achieve better separation. In this exercise, the length of DNA fragments will be estimated based on the sizes of marker bands and the information provided by your instructor.

Cleaning Up:

1. Return the 4X loading dye tube to the tube rack next to water bath. 2. Dispose the tips, sample tubes into big biohazard trashcan. 3. Transfer the gel running buffer into the recycling bucket. 4. Clean up the tank, the comb, the gel tray with tap water. Do not dry inside of gel box. Place the tray

inside the tank and make sure the lid matches the tank. 5. Refill yellow tip box. 6. Wipe your bench with 70% ethanol (clear top squirt bottle).


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Fig. 1: gel diagram Fig. 2: 500 bp DNA ladder

Fig. 3: The map of pGLO : The restriction site for EcoRV is at 386 and the sites for PstI are at 2106 and 3181)

PstI Double EcoRV Uncut pGLO

ladder EcoRV Double PstI ____ ____

5371 bp

wells

1000bp

500bp

1500bp 2000bp


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Post Lab Report (20 pts total): Include questions and a title page in your report (1 pt). 1. The restriction digest map of pUC19 is shown in Figure 4. Suppose you performed restriction digestions of pUC19 by the various enzymes listed in Table 1 (each row represents a separate digestion),

1) predict the sizes of fragments in base pairs and write your answers in Table 1(1pt); 2) Label the positions of the wells (1pt), label the lanes with the enzymes used (2pt). Include a lane

with 500 bp DNA ladder (1pt, Fig 2). Draw the corresponding DNA fragments on the gel diagram, and label the size of each DNA fragment (2pts). Label the cathode and anode (1pt).

Figure 4. Restriction map of pUC 19, a 2,686 base pair plasmid. The number after each restriction enzyme name indicates at which base pair the DNA is cut by that enzyme. Table 1.

Enzymes Fragments produced (bp)

EcoRI

Bsey I

Ava II

EcoR1 + Bsey I

EcoR1 + Ava II

Bsey I + Ava II

pUC19 2686 bp

0 EcoR I 396

Ava II 2059

Ava II 1837

Bsey I 1110


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2. Print the photograph of your gel from eLearning. Label the wells, the lanes with the enzymes

used (2pt). A 500 bp ladder containing 16 bands in 500bp increments (the smallest one is 500 bp) was used to estimate the sizes of DNA fragments on your gel. Note the sizes of the four smallest DNA fragments in the molecular ruler/ladder (1pt). Estimate and label the size of each fragment in each restriction digest sample (1pt). Calculate the sizes of fragments in base pairs based on the information provided in Fig 3 and label the size for each band (one set) (2pts). Label the cathode and the anode (1 pt).

3. Visit the New England Biolabs website (www.neb.com) and click on “NEB CUTTER” at the bottom of the screen. Open the file of pGLO sequence found at eLearning. Copy and paste the sequence into NEBcutter. Make sure the sequence doesn’t have linebreaks in it. Type “pGLO-your name-BIOL2281” in “Name of sequence”. Select “the sequence is circular”. Select NEB enzymes and click submit. You will get the restriction map for pGLO.

Then click on Custom Digest under Main Options. Choose EcoRV, PstI. Select “Digest”. It

will display the restriction map with just these enzymes. Click “view gel” under “Main options”. Print the page with a virtual gel and the list of fragments. Your name should be displayed on the title of the print-out (2pts).

On the page of “Custom Digest”, five of the open reading frames are identified on the plasmid map. Click on the curved bars of c. Click “Blast this sequence at NCBI” under the protein sequence and find out the name of the protein superfamily (1pt). Repeat the search steps for curved bar of b and find out the name of the protein superfamily (1pt).

http://www.neb.com/

BIOL2281 E5

Dr. Wenju Lin, BIOL2281_Spring 2016 1

Experiment 5: Restriction Enzyme Digest and Plasmid Mapping

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Restriction Enzyme Digest

Plasmid Mapping

Gel electrophoresis

Restriction Enzyme Digest

A strategy for obtaining fragments of DNA

Restriction enzymes cleave segments of DNA from the genome of various types of cells or fragment

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the genome of various types of cells or fragment DNA obtained from other sources

Restriction fragments of DNA from different sources can be used to synthesize a recombinant DNA

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DNA cloning: The new plasmid can be introduced into bacterial cells that can produce many copies of the inserted DNA.

http://www.accessexcellence.org/RC/VL/GG/inserting.html

Types of nucleases

Nucleases: catalyze the hydrolysis of phosphodiesters in nucleic acids

Exonucleases: from one end of a polynucleotide chain

Endonucleases: at various sites within a polynucleotide chain

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chain

Restriction Endonucleases (Enzymes):synthesized in some bacteria to protect against viral attack by destroying foreign DNA

How do they protect their own DNA? The host cell modify the bases of potential restriction sequences by methylation of adenine or cytosine

Nuclease cleavage sites

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Restriction recognition sequences

Restriction Enzymes (RE) cleaves the DNA within or near to the specific recognition sequences

the nucleotide sequences recognized by more than 200 RE can be classified as:

a. Tetra- or hexapalindromic sequences: they are the same sequences when read in the5' > 3' di ti f b th t d

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5' ---> 3' direction of both strands. Dpn I 5’GATC3’ EcoR I 5’GAATTC3’

3’CTAG5’ 3’CTTAAG5’

b. Pentanucleotide sequences.c. Sequences of longer extention with internal (N) sequences.

Xmn I (GAANNNNTTC)d. Nonpalindromic sequences.

(Nawin Mishra,2002)

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Sticky and Blunt ends

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Figure 8.3

Essential components of a restriction digest reaction

DNA plasmid

Appropriate buffer (10X)

dH2O

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dH2O

Enzyme (unit/µl) : the volume of enzyme should not be included in final reaction volume

One unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete digest on 1µg of substrate DNA in 60 minutes at the appropriate assay temperature in a 50µl reaction volume. Most restriction enzyme reactions are incubated at 37°C for one hour.

Microscale

The volumes and tools are adapted to microscale when we work with DNA

1 ml = 1000 µl (1000 microlitres)1 mg = 1000 µg (1000 micrograms)

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Microcentrifuge tubes

0.25 ml to 2.0 ml

Made of Polypropylene

With Flat tops for writing on

With Graduations, and writing area on the side of the tube.

Microcentrifuge

Use centrifugal force to separate lighter (rich medium) and heavier substances (cells).

Spin tubes to ensure efficient usage of

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Spin tubes to ensure efficient usage of valuable drops of reagent.

Use pause button for momentary spin

The load in a laboratory centrifuge must be carefully balanced.

Electrophoresis

a separation technique in which an electrical field causes charged molecules to move through a matrix (usually a gel).

routinely used to separate DNA, protein and

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y p , pother polymeric molecules.

Separation can be based on Sizes (DNA fragments separated by size).

Net charges shapes

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Electrophoresis Equipments & Setup

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1. The flat-bed tank

2. The gel tray

3. The comb

The Equipments The tray

The actual mold which provides a shape for the gel .

The comb: It is placed into slots in the tray, with the "teeth" down, when the

agarose is still hot. The agarose polymerizes with small "wells“ into which samples are added.

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The tank Holds the running buffer, the same buffer used to make the gel

is also used as the running buffer.

One power connection lead is positive (red) and one negative, there will be a strong electrical current flowing through the tank when the electrodes are immersed. The gel will be completely submerged as it is run.

Agarose gel electrophoresis

Agarose (nontoxic polysaccharides) gel Mixed with gel running buffer Melted Poured into a casting tray

DNA f t h d th ill b d t d th

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DNA fragments are _____ charged, they will be drawn toward the _____ electrode (anode,red). DNA fragments are separated by _______.

DNA fragments stained by ethidium bromide (EB) are visible using UV lamp. EB Intercalates into the DNA molecules and is a carcinogen.

The lengths of the DNA fragments can be determined by comparing the migration distances (mm) to the standard curve (log bp vs. migration distance) of the DNA ladder.

Visualizing DNA by Ethidium Bromide

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1 g/ml of EB included in the agarose gel or TBE buffer

500bp DNA ladder

Contains 16 bands in 500bp increments; the smallest one is 500 bp

Used to estimate DNA

wells

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Used to estimate DNA fragment sizes on a 0.8% -1.0% agarose gel.

The mobility of linear DNA fragments is inversely proportional to the log10 of their molecular weight.

500 bp

1000 bp

Effect of DNA Conformation on Mobility

The size of uncut plasmid DNA can not be accurately determined. Uncut plasmid DNA has several distinct conformations.

Ci l f li DNA

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Circular form vs linear DNA

Supercoiled vs nicked circles;

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Agarose gel electrophoresis (con.)

The total size of the plasmid can be obtained by adding up the size of

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obtained by adding up the size of each DNA band in one lane

Partial digests

Intensity of DNA bands correlate to the amount or the molecular numbers of DNA

Use of Restriction mapping

A description (roadmap) of restriction endonuclease cut sites within a region of DNA

To characterize an unknown DNA prior

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To characterize an unknown DNA prior subcloning or further manipulations, ex: recombinant plasmid mapping

DNA fingerprinting, ex: restriction digestions on PCR products, RFLP

Plasmid Mapping

Cutting a plasmid converts a circular molecule into a linear one.

Cutting a plasmid with two enzymes results in two linear fragments.

Enzyme 1

21How about cutting a linear PCR fragment?

Enzyme 2

Single digests and double digests

Single digests are used to determine which restriction sites are in the unknown DNA or to determine the size of a plasmid.

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Double digests are used to order and orient the fragments correctly.

Predict fragment size based on plasmid map

Single digest EcoRI- 2686bp Bsey I Ava II

0EcoR I 396

Ava II 2059

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Double digest EcoRI + Bsey I

1110-396=714, 2686-714=1972

EcoRI + Ava II Bsey I + Ava II

pUC 19 2,686 bp

2059

Bsey I 1110

Ava II 1837

Draw what you would see on a gel with the predicted fragments

Negativeelectrode

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DNA Marker

EcoR I

EcoR I+Bsey I

2686

1972 714

well 500 bp

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The map of pGLO 386

5371

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The restriction site for EcoRV is at 386 and the sites for PstI are at 2106 and 3181)

31812106

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What factors influence the rate of migration in DNA agarose gel?

Molecular size of the DNA The conformation of DNA

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The agarose concentration The buffer The applied voltage

The concentration of an agarose gel allows for the separation of different sizes of DNA fragments.

0.5% gel : providing better separation for fragments larger than 10 kb

1% gel: providing better separation for fragments

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gbetween 500bp-10kb

2% gel: providing better separation for fragments smaller than 1 kb

0.8% gel will be used in the lab

Voltage

The higher the voltage, the faster the rate of migration. However, Accompanying heat may melt low-percentage gel.

(Don’t use more than 130 volts)

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(Don t use more than 130 volts).

Imperfections in the gel distort the bands and produce ambiguous results (slants and smiles).

Loading Dye

Used to monitor the movement of DNA fragments, it does not stain DNA

TBE loading dye contains Glycerol Bromophenol blue: migrates as a 300bp fragment.

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Bromophenol blue: migrates as a 300bp fragment. Xylene cyanol: migrates as a 9000bp (9Kb) fragment.

10X stock routinely added to the samples beforeloading

In the lab: 15µl of reaction mix+ 5µl of 4X loading dye

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In the lab

Complete restriction enzyme digests of pGLO

Set up agarose electrophoresis

Complete Question1 in report E5

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Photo of gels will be posted on eLearning

Analyze the plasmid further at home by visiting the New England Biolabs website and NCBI website

Restriction fragments cut with two enzymes with complementary tails

Sal I

G* TCGA C G TCGAC

C AGCT *G CAGCT G +

G

CAGCT

TCGAC

G

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XHO I

C* TCGA G C TCGAG

G AGCT *C GAGCT C +

C

GAGCT

TCGAG

C

pre-lab e 5: restriction enzyme digest and plasmid mapping ... · biol 2281, spring 2016 e 5:...

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