RNA electrophoresis!!

Christian Stöckigt cstoeck1 at gwdg.de
Fri Oct 31 12:32:24 EST 1997



Han wrote in article <345A61CC.5290 at hotmail.com>...

>Dear netters,
>
>I encountered a problem in RNA formaldehyde electrophoresis: I added EB
>in my formaldehyde agarose gel(final concentration 50ug/100ml) and run
>gel for 2 hours. Amazingly, when I visualize gel under UV lamp I found
>very high staining background which evenly distributed and composed
>almost 2/3 area of gel starting from the margin near the wells. This high
>flurescent staining is so strong that overhided my RNA bands. I would be
>very appreciate any suggestion about the possible reasons. My RNA
>formaldehyde electrophoresis buffers are as following: (please save me
>out of the nightmare)
>
>10xMOPS:
>        MOPS  41.2 g
>  Sodium acetate 6.57 g
>  EDTA,sodium salt  3.7 g
>  water to 1 litre(pH to 7.0)
>
>1xMOPS gel running buffer: add EB to final concentration 1ug/100ml
>
>1% formaldehyde agarose gel:
> 37% formaldehyde 8.5 ml
> RNase free water 37.5 ml
> 10xMOPS 5 ml
> Agarose 0.5 ml
> EB 25 ug
>
>RNA loading buffer:
> RNA (10-20 ug) 5 ul
> 10xMOPS 1 ul
> 37% formaldehyde 3.5 ul
> formamide 10 ul
>
>
>Thanks in advance.
>
>HZ
>Gut hormone Lab
>K.U.Leuven

Hallo!

This problem seems to occur quite often. It happened to me as well..... Try
to stain  your gel after run in Na- Acetate (RNase free) containing EB. Do
not add EB to your gel. For me it serves very well!

Christian

Christian D. Stoeckigt
Center Internal Medicine
Dept. Gastroenterology/Endocrinology
Georg- August University
Robert- Koch- Str.40
37075 Goettingen, GERMANY
Tel.: ++ 49 551 39 6366
Fax:  ++49 551 39 8279
email: cstoeck1 at gwdg.de







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