发信人: Cajal (chump), 信区: Biology
标 题: Re: DNA难溶解会是什么原因呢？
发信站: BBS 未名空间站 (Tue Jul 19 19:27:49 2005)

I assume your template has already have sp6/T7 or whatever promoter that is ne
eded for in vitro transcription. Then try this one:
In short, gel purify your RNA probe instead of your DNA template.

You don't necessarily need to gel purify the template. What we do is to linear
ize the template, then ethanol precipitate the linearized template. I assume y
ou know how to EtOH precipitate nuclear acids. The key is when you air dry the
pellet, don't let it become too dry. Then you dissolve the pellet in RNase f
ree water, spect your solution to get the concentration, do the in vitro trans
cription.
After transcription, run all your reaction on 5% denaturing urea gel, do autor
adiography to locate your RNA probe band. (You should be able to see your prob
e after <5 min exposure on X-ray film. ) Then cut that band out, extract the g
el slice with 0.5 ml buffer X for 45-60 min, add 2 ul of 30 mg/ml glycogen to
the supernatant, precipitate with 1 ml EtOH.

buffer X:
1 ml 5M NH4Ac (Ammonium Acetate)
0.1 ml 500 mM EDTA
0.5 ml 10% SDS
Add water to 10 ml, adjust pH with 10M NaOH to 7.0-7.5

5% denaturing gel:
0.5 ml 10X TBE
0.85 ml 30% Bis-Acrylamide
1.3 ml deionized formamide
2.4 g urea
add water to 5 ml.
5 ul TEMED, 50 ul APS

BTW. even you don't have to gel purify your DNA template, you may still wanna
think about firing your PI. The Qiagen gel extraction kit costs $76 for 50 pre
ps. My PI does not have funding either, yet he is never cheap on reagents.

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