Download Agarose Gel Electrophoresis

HHMI Undergraduate Research Studio - Freshmen Biology Section, Fall 2007
Agarose Gel Electrophoresis
- Agarose:
- linear polymer composed of alternating isomers of the sugar galactose (D- and Lgalactose)
- polymers aggregate to form supercoiled structures of a radius of 20-30 nm and variable
length (around 800 galactose residues)
- standard agaroses melt at ~ 90 ºC and gel at ~ 40 ºC; gelation results in mesh of
channels with diameters from 50- >200 nm (see figure)
Figure: Gel structure of agarose
- Migration and separation of DNA in agarose gels:
- DNA is negatively charged and therefore migrates to the anode (positively charged
electrode), if a voltage is applied
- rate (speed) of migration depends on:
- size of the DNA (length in base pairs, bp, or kilobase pairs, kb): large DNAs
migrate slowly (because it is more difficult for them to wiggle through the pores),
whereas small DNAs migrate faster (because they can easily penetrate the pores;
you can think of the gel as a “sieve”); as a result, DNAs are separated by size and
the size of a DNA fragment can be determined by letting standard DNA fragments of
known size run in parallel to the experimental samples [for those who want to know
the precise relationship between rate of migration and size: the rate is inversely
proportional to the log10 of the number of base pairs in a DNA fragment]
- agarose concentration: the lower the concentration, the faster the DNA fragments
will migrate; in general, if the goal is to separate large fragments, a low concentration
of agarose should be used (e.g., use 0.7 % agarose if you want to separate a 3-kb
fragment from fragments of similar size); if the goal is to separate small fragment, a
high concentration of agarose should be used (e.g., use 2 % agarose if you want to
separate a 100-bp fragment from fragments of similar size)
- conformation of the DNA: the relationship between rate of migration and size of
the DNA explained above only applies to linear DNA; different forms of circular DNA,
e.g. undigested plasmid DNA, have different properties and will migrate with different
rates as the same DNAs in their linear form; plasmids and other circular DNAs must
therefore be linearized by restriction digestion before running them on a gel with the
goal to determine the size of the DNA
- voltage applied: the higher the voltage, the faster the DNA will migrate; do not
exceed 5-8 V/cm; it is best to check the current and make sure that it not exceeds 75
mA for standard size gels and 100 mA for minigels; heat generated at higher
voltages/currents will impair the resolution or even melt the gel
- buffer composition: the salt content (ionic strength) of the electrophoresis buffer
will influence migration; without salt the DNA will barely move; in the presence of two
much salt, the conductivity will be very high and the produced heat will impair
separation; we will use a Tris-borate buffer (pH 8.0) that contains EDTA (TBE); EDTA
is a chelating agent that binds divalent cations such as Mg++ that many nucleases
require for their activity; EDTA thus protects the DNA from enzymatic degradation
- Gel loading buffer, added to DNA sample:
- contains glycerol or sucrose to increase density (otherwise, sample would dissolve in
running buffer and not sink into the gel pocket);
- contains dyes that facilitate observation of the sample during gel loading and
electrophoresis:
- bromophenol blue: runs about as fast as 300-bp DNA (linear!); separation is usually
stopped shortly before this dye reaches the end of the gel (depending on the size of
the fragments that need to be recovered)
- xylene cyanol (optional): runs about as fast as 4-kb DNA
- Ethidium bromide staining of DNA:
- Ethidium bromide is a fluorescent dye that binds to DNA (intercalates between the
stacked bases); binding increases fluorescence about 10-fold
- when irradiated with UV light of a wavelength of 302 nm, ethidium bromide will emit
fluorescence light of a wavelength of 590 nm (orange)
- the dye can be included in both the running buffer and the gel, the gel alone, or the
gel can be stained after DNA separation (which comes in handy if you forgot to add
the dye before running the gel); to minimize ethidium bromide-containing waste, we
will include the dye only in the gel at a concentration of 0.5 mg/ml (added to the buffer
used to dissolve the agarose; disadvantage: ethidium bromide is positively charged
(see structure) and will migrate in the opposite direction as the DNA, which leads to
an uneven background staining)
- detection limit: ~ 10 ng DNA (detection can be improved by soaking the gel in H2O
for ~ 30 min to reduce background staining)
- Achtung: ethidium bromide is a strong mutagen (does that surprise you?) and
should be handled with care (always wear gloves, avoid skin contact; if contact
occurs, wash immediately with plenty of water); ethidium bromide-containing gels will
be treated as hazardous waste (special container)
Structure of ethidium bromide