For separating DNA fragments between 600 bp and 5 kb we use 1% agarose
gels. Higher resolution in the 300 bp - 1 kb range can be achieved using 1.5%
agarose.
We have different grades of agarose for different applications. For analytical
work use BioRad LE or BioRad high strength analytical grade or agarose from
BioShop.
Preparing Stock Solutions
50X TAE Stock:
TAE is: Tris Acetate EDTA
1X concentrations
50 X amounts
Tris base pH 8.5
.04 M
242 g
glacial acetic acid
.002 M
57.1 ml
EDTA pH 8.5
.002 M
37.2 g
Pouring Agarose Gels
Dilute 50X TAE stock to 1X
Set up the agarose gel electrophoresis system with combs as illustrated in the Bio-Rad Sub-Cell® GT
Agarose Gel
Electrophoresis System Instruction Manual:
In a glass flask add the required amount of agarose to 1 X TAE. For 100 mls of
1% agarose, add 1 gram to 100 mls of 1 X TAE.
Melt the agarose by placing flask in the
microwave and bringing to a boil. You have to stand and watch it or it will boil over and make a mess.
For analytical agarose bring to a boil a
couple of times, swirl in between and that should do. GTG agarose is harder to melt
Warning: When using a microwave to melt agarose you often heat it several times.
This can cause the solution to become super-heated and then when you swirl it -
it will boil all of a sudden and can spill agarose out of the top of the flask
and burn you. Since it sticks to your skin you can get a nasty burn unless
you are wearing waterproof insulated glove
After it is completely melted allow it to cool until it is warm but not hot to the touch
(approx 50oC). Pour the agarose into the electrophoresis system.
Allow agarose to solidify for 1/2 hour to 1 hour depending on size.
Pull out combs.
Fill electrophrosesis system with 1X TAE buffer.
Running Agarose Gels
Program gel electrophoresis system to run at 140V for approximately 1.5 hours.
After you have run your gel, cut out the piece you have used and place it
in
~100 mls of TAE buffer containing ethidium bromide for 15- 25 minutes. Ethidium bromide is light
sensitive (especially to fluorescent lights) so keep the lid on the
canister.
Ethidium bromide is stored at 4° C in solution at 5 mg/ml. It is a mutagen in vitro
and therefore probably also in vivo. Therefore, handle ethidium bromide solutions with
caution. Wear gloves at all times. To calculate the correct volume to add, divide your
total buffer volume in mls by 20. The result is the number of ul of 5 mg/ml stock to add.
For our example, 100 mls / 20 = 5 .. add 5 ul. Add this same amount to any solution
used for staining DNA.
Recovering DNA from Agarose
For recovering DNA
from agarose pour a new gel and do not use stock buffers as they are likely
growing bugs.
If you are having recovery problems try using GTG agarose. SeaKem GTG is used for DNA
fragments larger than 300 bp. For smaller fragments use Nusieve GTG. To
get your DNA back you can electro-elute or just melt it and use it melted. Nusieve GTG is a low melting point agarose
which once melted will stay liquid at 37°C. Nusieve is very expensive so use it only for
applications where other agaroses will not work. Similarly, GTG agarose is 2-3 times
more costly than analytical agarose.
Use one of the matrix binding methods (diatomaceous
earth - Prepagene matrix from BioRad; Ground porcelain - Glassmilk from Bio101) for preparative work
with fragments >300 base pairs.
For fragments <300 base pairs use this method.
