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DNA gel electrophoresis

For DNA gel electrophoresis, a gel is cast from

agarose, dissolved in buffer solution. Agarose is a very

pure (and expensive) form of agar, which is obtained

from seaweed. At one end of the slab of gel are several

small wells, made by the teeth of a comb that was

placed in the molten agarose before it set. A buffer

solution is poured over the gel, so that it fills the wells

and makes contact with the electrodes at each end of

the gel. Ions in the buffer solution conduct electricity.

The test samples (DNA fragments) are mixed with a

small volume of loading dye. This dye is dissolved in a

dense sugar solution, so that when it is added to the

wells, it sinks to the bottom, taking the DNA sample

with it. An electrical potential is applied across the

gel. Phosphate groups give the DNA fragments a

negative electrical charge, so that the DNA migrates

through the gel towards the positive electrode. Small

DNA fragments move quickly through the porous

gel; larger molecules travel more slowly. In this way

the pieces of DNA are separated by size. The loading

dye also moves through the gel, so that the progress

of the electrophoresis can be seen (the DNA itself

is invisible).

Copyright © NCBE, University of Reading, 2017

NCBE BRIEFINGGEL ELECTROPHORESISGel electrophoresis is a key technique in modern biology that features in all of the

new A-Level Biology specifications in England. It is a way of separating DNA, RNA or

proteins based on their size and/or the electrical charge on the molecules.

Visualising DNA

After electrophoresis, the DNA is visualised. In

research laboratories, a fluorescent dye will have

been incorporated into the agarose gel before it

was cast. After the gel has been ‘run’ it is illuminated

with ultraviolet (UV) light and the dye, which binds to

DNA, shows up as bright fluorescent bands. Ethidium

bromide was until recently the most commonly used

DNA stain. Ethidium bromide has similar dimensions

to a base pair in DNA. When ethidium bromide binds

to DNA, it slips between adjacent base pairs and

stretches the double helix. This causes errors when

the DNA is replicated.

No. 1 | JANUARY 2017

Short-wavelength UV light is itself harmful and

ethidium bromide’s breakdown products are thought

to be potent mutagens and carcinogens. Ethidium

bromide should therefore not be used in schools*.

For reasons of safety and because UV light of this

wavelength causes unwanted mutations in the DNA

being studied, several alternative stains are now

often used in research labs. These include SYBRsafe®

and GelRed®, which although they are thought to be

safer than ethidium bromide, are far more expensive

[Ethidium bromide costs £4.50 per mL compared

with £133 per mL for SYBRsafe® and £200 per mL for

Above: Intercalation of ethidium bromide between two adjacent bases in a DNA molecule.

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GelRed® (2016 prices).] An additional advantage of

some of these compounds is that they will fluoresce

in blue, rather than harmful UV light.

In schools, safer, cheaper dye solutions are used

to stain the entire gel, including the DNA, after

electrophoresis. Suitable stains include Azure A and

Azure B, Toluidine blue O and Nile blue sulphate. This

type of stain is not thought to intercalate within the

DNA double helix, but instead binds ionically to the

negatively-charged phosphate groups of the DNA.

Such dyes are not as sensitive as ethidium bromide

and the newer fluorescent dyes, and some of them

may colour the gel heavily. Consequently, prolonged

‘destaining’ in water may be necessary before the

DNA bands can be seen. Methylene blue, which is

sometimes used for staining DNA on agarose gels in

schools, is far from ideal, as it requires destaining and

it fades rapidly after use.

Although these alternatives to ethidium bromide

are thought to be relatively safe, they have not been

intensively studied for long-term effects and the

mechanisms by which they bind to DNA are not fully

understood. As with all laboratory chemicals, suitable

safety precautions should be exercised when handling

any dyes, particularly when they are in dry, powdered

form.

Viewing gels

Gels stained with a blue dye such as Toluidine blue O

can be viewed in daylight. A smartphone with a white

background light (some ‘torch’ apps are suitable) can

be used as a ‘lightbox‘. Alternatively, LED lightboxes

sold for tracing can be used. A yellow-coloured filter

may help to enhance the contrast when photographing

gels that have been stained with blue dyes.

Storing stained gels

Gels stained with Toluidine blue O or Azure A can be

stored refrigerated in a plastic bag to prevent them

from drying out. Provided they are not exposed to

light, gels kept like this will not fade for many months.

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Azure B

Azure A

Toluidine blue O

Nile blue sulphate

Above: Some dyes that are thought to bind ionically to DNA.

HOW MUTAGENIC ISETHIDIUM BROMIDE?

In recent years there has been some

controversy about the dangers of ethidium

bromide. The compound is used as a drug

for treating cattle with trypanosomiasis

(African sleeping sickness) at far greater

concentrations than are used in the lab. The

cattle do not seem to suffer any adverse

effects, but since the animals are usually

slaughtered after a few years, any long-term

harm would not be noticed. When ethidium

bromide is metabolised in the liver, the

compounds produced are highly mutagenic.

It is probably correct to say that ethidium

bromide is not as harmful as some people

think it is, but it should still be handled with

care and disposed of correctly. The relevant

safety regulations state that it MUST NOT be

used in UK schools*.

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Ethidium bromide

* See: www.ncbe.reading.ac.uk/SAFETY/dnasafety.html

3 www.ncbe.reading.ac.uk

No. 1 | JANUARY 2017

Copyright © NCBE, University of Reading, 2017

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What’s the best voltage to use?

At low voltages, movement of linear DNA is proportional to the voltage

applied. As the voltage is increased, the mobility of the higher molecular

mass fragments is increased differentially (the larger fragments tend to

‘catch up’ with the smaller ones). Hence the effective range of separation

is decreased as the voltage is increased. For the best resolution, 0.8%

agarose gels should be run at no more than 5 V per cm (as determined

by the distance between the electrodes). The NCBE electrophoresis

equipment, which is designed to work at 36 V, has a distance between the

electrodes of ~85 mm, which is close to the optimum.

Calculating the resolution of a gel

For λ DNA digested by HindIII (shown on the left), the resolution can be

calculated by dividing the distance between the 23 and 2 kb fragments by

the total distance travelled by the 2 kb fragment.

Fragmentsize (kb)

5 V / cmGood

separation

Gel concentration also affects the movement of DNA fragments. There

is a linear relationship between the logarithm of the mobility of the DNA

and the gel concentration. By altering the agarose concentration it is

possible to control the range of sizes of fragments that can be separated by

electrophoresis. The example on the left shows λ DNA digested by HindIII.

The optimum gel concentration for separating these λ DNA fragments is

~0.8% (w/v), which is the concentration suggested in the NCBE’s Lambda

protocol module (see page 6).

For larger or smaller DNA fragments, a different agarose concentration

may be better. For instance, to show chloroplast DNA fragments of

between ~500 and several thousand base-pairs, such as those produced

by the NCBE’s PCR and plant evolution module, an agarose concentration

of 1.5% (w/v) is recommended. The table below shows the concentration

of agarose needed for separating DNA fragments of different sizes.

0.5 1.0 1.5 2.0

Gel concentration (%)Agarose(% w/v)

Separation range(kb)

Relativegel strength

0.3 60–5 Very weak

0.6 200–1 Weak

0.7 10–0.8 Moderate

0.9 7–0.5 Moderate

1.2 6–0.4 Strong

1.5 4–0.2 Strong

2.0 3–0.1 Strong

EFFECT OF VOLTAGE

EFFECT OF GEL CONCENTRATION

Above: Even a small difference in gel concentration can have a significant effect on the quality of the results you see. For that reason, it is important to make up agarose solutions accurately, using buffer, not water. Don’t try to weigh out small amounts of agarose, make up a large volume: it will keep indefinitely in a sealed container.

20 V / cmPoor

separation

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Polyacrylamide gels and protein electrophoresis

To separate proteins by electrophoresis, gels cast

from polyacrylamide are sometimes used. Before

proteins are run on a gel, they are treated with a strong

detergent, sodium dodecyl sulphate (SDS). This,

coupled with heating, causes the tightly-folded protein

molecules to unfold and become linear, so that they

will move through the gel according to their sizes, not

the way in which they are folded. The SDS also binds to

the proteins, giving them an overall negative charge,

so that they move towards to positive electrode. This

type of electrophoresis is called SDS-PAGE (SDS-

polyacrylamide gel electrophoresis).

It is also possible to separate proteins using a special

type of agarose, but in contrast to the procedure

using polyacryamide, with agarose the proteins are

separated by electrical charge only (not charge and

size). This is because the pores within the agarose

gel are relatively large and the proteins can easily pass

through them.

As with DNA, the proteins on the gel are stained

with an appropriate dye. Dyes originally developed

for textiles such as Coomassie blue (which bind

to proteins like wool) are often used. De-staining

(often with water) is then necessary to remove the

background stain from the gel before the protein

bands can be seen.

SAFETY OF POLYACRYLAMIDE GELSPolyacrylamide gels must not be cast in a school, as the two materials used to make them (acrylamide and bis-acrylamide) are neurotoxins. Safe, pre-cast polyacrylamide gels may be purchased, but it is important to check their shelf-life, as they can seldom be stored for more than 12 months.

Restriction enzymes

Whole genomic DNA is too big to run on a gel.

Typically, one or more restriction enzymes

(restriction endonucleases) are used to cut the

DNA molecules into smaller fragments before

electrophoresis. Such enzymes are produced by

bacteria as a defence against ‘foreign’ nucleic acids

e.g., from invading bacteriophages. These enzymes

bind to specific sequences of bases in double-

stranded DNA and cut the DNA, either directly at

the sites they 'recognise' and bind to, or at another

position in the DNA molecule. Small differences in

DNA sequences that can be detected by the action

of such enzymes are called ‘restriction fragment

length polymorphisms’ (RFLPs). These are often

used as genetic markers when they occur near to

genes of interest that are difficult to detect directly.

Above: Restriction enzyme BamHI bound to double-stranded DNA. This view is looking down the axis of the DNA molecule (ball-and-stick model, in the centre of the image). The restriction enzyme is shown in ‘cartoon‘ format, with b-pleated sheets in yellow and a-helicies in magenta.

This image uses data from: Newman, M., et al (1995) Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663 [Protein Data Bank ID: 1BHM]. The software used to produce the image was UCSF Chimera and VMD, which can be obtained from: www.cgl.ucsf.edu/chimera/ and: www.ks.uiuc.edu/Research/vmd/ respectively.

Restrictionenzyme nameSource microorganism STRAIN

DNA base pair ‘recognition’ site (5'a3')

BamHIBacillus amyloliquefaciens H

G$GATCC

EcoRIEscherichia coli RY13 G$AATTC

HindIIIHaemophilus influenzae Rd A $AGCTT

Above: Some examples of restriction enzymes.

5 www.ncbe.reading.ac.uk

No. 1 | JANUARY 2017

NCBE ELECTROPHORESIS PRODUCTS

The NCBE’s award-winning electrophoresis

equipment is probably the world’s the most

cost-effective system for gel electrophoresis.

More than half a million sets have been provided

to schools since 1992. The NCBE’s prototype

electrophoresis kit is now in the Science Museum

in London.

The NCBE equipment uses very little agarose and

buffer, making it economical to run.

There are three parts to the NCBE’s electrophoresis

system. All of the re-usable items (gel tanks, combs

etc.) come in a BASE UNIT. The base unit contains

eight sets of equipment.

To power the electrophoresis equipment, we supply

a 36 V MAINS TRANSFORMER. This is a safe, fast

and economical alternative to the batteries that

some people have used in the past.

Finally, all of the consumable items (agarose, DNA,

enzymes etc.) are provided in MODULES. The

modules’ contents vary, but they usually include

sufficient materials for 16 students or working

groups to carry out the practical work. Full details

are given on the NCBE web site: www.ncbe.reading.

ac.uk/electrophoresis.

All of the module contents will keep, if stored

correctly, for at least a year.

Copyright © NCBE, University of Reading, 2017

How do I decide what I need?

Decide how many base units you need, according to

your class and/or working group sizes. Remember

that the base unit contains eight sets of hardware.

Next, choose which module(s) you’re interested

in. Again, you’ll need to order the correct number

for your class size(s). The modules also act as

‘refill packs’, although you can also buy most items

individually.

Electrophoresis base unit

This pack contains eight sets of the items required

for gel electrophoresis.

8 NCBE gel tanks; 8 4-toothed combs; 8 6-toothed

combs; 8 pairs of red and black wires with crocodile

clips; 8 microsyringe dispensing units (without tips).

36 volt mains transformer

This transformer is a safe, cost-effective

and environmentally-friendly alternative to

batteries. With the connector provided, a single

transformer can power four NCBE gel tanks.

At 36 volts, the ideal voltage for the NCBE

electrophoresis equipment, a 0.8% agarose gel

will take two hours to run: gels made with a greater

concentration of agarose may take slightly longer.

Individual replacement items

The page overleaf describes the ‘modules’ of

consumable items for gel electrophoresis that the

NCBE currently supplies. All of the individual items from

these modules are also available separately.

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NCBE ELECTROPHORESIS MODULES

Nature’s dice

Genetics is often difficult for students to

understand. This innovative practical work uses

modern DNA technology to help students learn

about classical Mendelian inheritance.

This exercise provides a practical simulation

of genetic screening, centred on a fictitious

extended family with 24 members. The DNA

samples can be distributed by the teacher so that

students can investigate the inheritance of either

a sex-linked or an autosomal recessive condition.

Students treat the DNA samples provided

with a restriction enzyme and run them on

electrophoresis gels. The results from the class

are pooled so that the pattern of inheritance may

be determined. This activity is a novel practical

way of reinforcing learning about Mendelian

inheritance, the use of restriction enzymes and gel

electrophoresis. It presents an ideal opportunity

to stimulate discussion about genetic counselling,

confidentiality of genetic information and other

ethical concerns.

The lambda protocol

This practical exercise has become a classic for

demonstrating the action of different restriction

enzymes on DNA.

The bacteriophage lambda (λ) has double-

stranded DNA which is 48,502 base-pairs in length.

Different restriction enzymes ‘recognise’ specific

sequences of bases in this DNA and cut it at precise

locations. Three different restriction enzymes are

provided in this module: BamHI, HindIII and EcoRI.

After treatment with the individual enzymes,

the lambda DNA fragments are separated by gel

electrophoresis. Once the gel has been run, the

DNA is stained to reveal distinctive patterns of

bands which correspond to fragments of different

sizes.

The PCR and plant evolution

This module allows students to amplify chloroplast

DNA using the polymerase chain reaction (PCR).

The length of the fragments produced can be used

to infer evolutionary relationships.

The polymerase chain reaction (PCR) is one of

the most important and powerful methods in

molecular biology. It enables millions of copies of

specific DNA sequences to be made easily and

quickly. The technique and variations of it are used

extensively in medicine, in molecular genetics

and in pure research. This practical kit provides

materials for the simple extraction of chloroplast

DNA from plant tissue, its amplif ication by the

PCR, and gel electrophoresis of the PCR product.

Students can use plants of their choice and identify

possible evolutionary relationships between

different species. This mirrors the molecular

methods used in modern plant taxonomy. This

activity presents an ideal opportunity for open-

ended investigations by individual students or

groups.

Protein power!

The NCBE’s electrophoresis equipment can be

used to analyse proteins as well as DNA. You do,

however, need a special type of agarose (which is

supplied in the box) to carry out this work.

Small samples of protein-containing foods (e.g.,

fish or nuts) are mixed with Laemmli buffer. This

linearizes the proteins and gives them a negative

electrical charge. The samples are separated by

electrophoresis, then the gel is stained and de-

stained to reveal the protein bands.

National Centre for Biotechnology Education, University of Reading, 2 Earley Gate, Reading RG6 6AU. United Kingdom Tel: + 44 (0) 118 9873743. Fax: + 44 (0) 118 9750140. eMail: NCBE@reading.ac.uk Web: www.ncbe.reading.ac.uk