long run sequencing
bckraev at aeolus.ethz.ch
Fri Oct 21 15:18:01 EST 1994
After having played around a lot with gels, buffers etc I settled down with the
1. Bases 30-300/350 - a 30x50 cm 5% gel with "electrolyte gradient" ( 0.8 M
sodium acetate in the lower tank ), 3.5 hr at 55 W constant power, with two
2 mm thick alumina plates.
2. Bases 300-800(1000) - 5% Long Ranger, 30x80 cm, run at 70 W constant power
for 14-18 hr, with two 2 mm thick alumina plates.
Both gels are made on a so-called "high pH TBE" (10X: 162g tris-27g boric acid-
10g EDTA.2Na-salt, pH about 9), and have a thickness of 0.4 mm. The gel is
always made on 1X HTBE, for short runs(only) the tank buffer is 0.5X HTBE.
I think it is important to use HTBE, since it has a higher buffering capacity.
I did try 4% gel, but it was somewhat difficult to handle, especially to insert
the shark tooth comb without distorting the surface. In my experience gels
lower then 5% seem to have a feature that may be described as "low capacity",
that is, you get sharp bands only when you load very little material in a
small volume, e.g., 1/5 of a regular sequencing reaction, while with my
favourite pair I load 1/2 on each( 4-5 ul) and can read the next day.
Good luck, Sasha.
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