loading RNA samples on denat. PAGE

Simone Marker via methods%40net.bio.net (by marker from rhrk.uni-kl.de)
Mon Jan 21 05:55:31 EST 2008


Hi,

I have problems to get uniform bands when I load RNA samples on a denaturing 
polyacryle amide gel:

My RNA samples are dissolved in formamide and my loading buffer is a 
standard buffer with formamide, EDTA and bromphenole blue. Running buffer is 
0,5x TBE.
I want to analyse samll RNA (23nt), so I have a 0,4mm thick 15% gel (like 
sequencing gels, denaturing agent is 7 M urea). I load 10 microlitres on the 
slots (its no problem to get uniform slots with adequate size).
The problem is, that the samples (at least the bromphenole blue) diffuse 
into the gel bars between the slots immediatly after loading. Besides, when 
I start the run the single samples enter the gel in different ways, 
resulting in lanes of different width. After staining the gel I can still 
see different width of the lanes and bands, although the slots were uniform. 
Furthermore, the bands are not sharp enough.
I run the gel at ca 1800-2000V (ca 50-60W) and 50°C.

Does anyone have an idea how to improve the running quality?

Thank you very much in advance,
Simone




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