Berglund:Method1: for complex gels

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Acrylamide/agarose gel mix

homemade 40% 80:1
39.344g acrylamide
0.4918g bisacrylamide
100ml water total volume
filter before using

10xTG (fc=50mM) 7ml
40% 80:1(fc=4%) 7ml
water 21ml
1%agarose/water fc=0.5% (slightlycooled) 35ml
APS(1g/10ml water) 320µl
TEMED(Biorad brand) 127µl


running buffer
1xTG
50mM Tris pH 8
50mM Glycine

This type of gel is used to look at splicing complexes A, B, and C. You use a sequencing rig and vertical plates of your choice in size (I prefer 8inch or shorter plates). The results look better than running the complexes on a agarose gel, but are harder to produce.
You have to work quickly to pour and get the combs in. The heat of the agarose causes the acrylamide to polymerize faster than normal and so good hands are required. Problems that I have seen with these gels are: 1) the gel has slid through the vertical plates when mounted onto a rig, especially short plates that are 30cm long 2) the gels are cranky and with the glycine being a poor buffer, you have to watch the temperature of the gel as it is running or you will snap glass plates quite dramatically. Gels need to cure at least 3 hrs at RT or overnight in the fridge before using.
Gels have to be dried afterwards and then exposed to STORM plates or X-ray film.

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