Agarose Gel Electrophoresis:
Mini-gel
(Gibco/BRL rig)
Large gel (Owl)
Run Gel Prepare 100 mls (Mini gel) or 200 mls (Large gel) of Agarose in 1x TBE
1% agarose is 1 gram of agarose in 100 mls of 1xTBE.
For fragments 0.5-5 KB
use 1% agarose
For fragments 0.1-0.5 KB use 1.3 % agarose
For fragments larger than 5 KB use 0.8% agarose
For uncut plasmids use 0.8% agarose
The agarose/buffer mixture must be heated to boiling to get the agarose into solution
Microwave agarose + TBE for 2 min per 100 mls.
Allow cooling to 55oC in water bath.
This is very important or you will warp and destroy the gel tray. For Mini-gel:
Assemble tray, dams in gel electrophoresis chamber
Use a 5 ml pipet to seal the dams with a bead of agarose.
Once the bead has hardened (~5 min) pour in agarose.
Add comb. For Large gel
Assemble tray in pouring stand
Once agarose has cooled to 55oC pour into tray.
Add comb.Add loading buffer to samples
Allow agarose to harden (30min- 1hour) then add TBE buffer.
Load samples
Run gel
Mini gel 50 100 volts
Large gel 80-125 volt.
Run until bromophenol blue is at bottom of gel.
Stain in TBE + ethidium bromide (2ul/300 mls of the 10mg/ml stock of EtBr.
Document gel.
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