BIG PROBLEM WITH NORTHERN BLOT

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Akaleesh
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BIG PROBLEM WITH NORTHERN BLOT

Post by Akaleesh » Tue Jul 22, 2008 8:25 pm

PLEASE HELP!!!!!!!

I've run an RNA gel and northern blot but accidentally run at against a DNA molecular weight marker. Does anyone know whether I can still use this marker to work out the size of the transcripts i have detected on the blot??????

THANKYOU SO MUCH IF YOU CAN HELP!!!
MUCH APPRECIATED!!!

MisterATP
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Re: BIG PROBLEM WITH NORTHERN BLOT

Post by MisterATP » Thu Jul 24, 2008 7:51 pm

I think if diferent markers (DNA and RNA) are usually used so there is something diference between whem. I advise you to repeat the blotting (right results are much more important).

snowcapk
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Post by snowcapk » Sun Jul 27, 2008 9:40 pm

Your RNA gel probably contained formaldehyde and you probably denatured your samples (including the ladder) with heat and formaldehyde before loading them onto the gel. DNA weight markers are generally double-stranded, but under these conditions the DNA MW marker would be single-stranded just like your RNA sample. The difference in weight/size per nucleotide is not very large between deoxyribonucleotides and ribonucleotides, so I'm guessing that your DNA weight marker would give fairly accurate results, except that the band for "500bp" would really be "500nt" because of the formaldehyde.

Experiments have to be repeated so eventually you will need to run a similar gel/northern blot. My advice is to load both DNA and RNA weight markers on that RNA gel, and then compare the ladders to each other. This will tell you how you should interpret the ladder from the first gel.

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