Download Sample newsletter January 2017

No. 1 | JANUARY 2017
NCBE BRIEFING
GEL ELECTROPHORESIS
Gel 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.
DNA gel electrophoresis
Visualising DNA
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.
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.
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).
Above: Intercalation of ethidium bromide between
two adjacent bases in a DNA molecule.
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
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Copyright © NCBE, University of Reading, 2017
NCBE BRIEFING
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.
Azure A
Azure B
Toluidine blue O
Nile blue sulphate
Above: Some dyes that are thought to bind ionically
to DNA.
HOW MUTAGENIC IS
ETHIDIUM 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*.
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.
* See: www.ncbe.reading.ac.uk/SAFETY/dnasafety.
html
www.ncbe.reading.ac.uk
Ethidium
bromide
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Copyright © NCBE, University of Reading, 2017
No. 1 | JANUARY 2017
EFFECT OF VOLTAGE
Fragment
size (kb)
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.
5 V / cm
Good
separation
20 V / cm
Poor
separation
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.
EFFECT OF GEL CONCENTRATION
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 (%)
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.
Copyright © NCBE, University of Reading, 2017
Agarose
(% w/v)
Separation range
(kb)
Relative
gel strength
0.3
60–5
Very weak
0.6
200–1
Weak
3
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
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NCBE BRIEFING
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 (SDSpolyacrylamide 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 GELS
Polyacrylamide 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 doublestranded 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.
Restriction
enzyme name
Source microorganism STRAIN
DNA base pair
‘recognition’ site
(5'a3')
BamHI
Bacillus amyloliquefaciens H
G$GATCC
EcoRI
Escherichia coli RY13
G$AATTC
HindIII
Haemophilus influenzae Rd
A $AGCTT
Above: Some examples of restriction enzymes.
www.ncbe.reading.ac.uk
Above: Restriction enzyme BamHI bound to doublestranded 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.
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Copyright © NCBE, University of Reading, 2017
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.
How do I decide what I need?
36 volt mains transformer
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.
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.
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).
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.
Copyright © NCBE, University of Reading, 2017
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NCBE BRIEFING
NCBE ELECTROPHORESIS MODULES
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 doublestranded DNA which is 48,502 base-pairs in length.
Different restriction enzymes ‘recognise’ specific
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
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
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.
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.
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.
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 amplification 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 openended investigations by individual students or
groups.
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 destained 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: [email protected] Web: www.ncbe.reading.ac.uk
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