RT PCR produces nice crisp bands but of wrong lenght :-)

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twomad
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RT PCR produces nice crisp bands but of wrong lenght :-)

Post by twomad » Tue May 15, 2007 11:00 pm

I would like to ask for an advice. I am trying to
amplify a gene (about 800 bp) for a purpose of
expression later on.

My idea was to extract the whole RNA, do RT PCR
with nested primer (about 150 BP around my gene
of interest). In the next step I wanted to use
the cDNA obtained for one more PCR to amplify the
gene, which I would then later subclone to an
expression vector.

The problem is that after RT PCR, for which I use
outer nested primers, I get a band of 500 BP
instead of expected 1100. The primers seem to be
unique enough to not to amplify some other
sequence.


I will greatly appreciate your suggestions.

david23
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Post by david23 » Wed May 16, 2007 2:18 am

ok to narrow down the problem, you sure the primers you selected were right right? Can you post the sequence and primer here?

It's not likely that your RNA got degraded cleaved or something I suppose.

twomad
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Post by twomad » Wed May 16, 2007 8:36 am

Yes, I think the primers were right. I attached a file with the sequence. I made it into jpg file cause the site only allows pictures. I hope it is readable.
Thanks for the help.
Attachments
sequence.jpg

david23
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Post by david23 » Wed May 16, 2007 4:01 pm

I dont think there is anything wrong with the primer choice. But I keep on thinking back to fact that something might have happened to your RNA. Is it possible that your RNA or cDNA got degraded in some way, and only one of the primer worked, and made a partial. gene sequence. I am just throwing ideas here.

blcr11
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Post by blcr11 » Thu May 17, 2007 1:16 pm

Is it possible that your source tissue expresses an alternatively spliced message? It could be spliced to express a shorter protein, but retain the same upstream and downstream sequences. Or it could be spliced to remove one of your outer primer binding sites and you end up amplifying only one strand of the message. In either case, the actual message has to be smaller than you expect in order to explain what you’re seeing. I guess I would check the RNA sample to see that the size of the message(s) is consistent with what you expect. Degradation is always a possibility with RNA, but it sounds like at least one of your outside primers is working, so maybe your RNA is OK.

twomad
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Post by twomad » Thu May 17, 2007 11:01 pm

david23, blcr11

Thank you for your time. There are no other know splicing variants of the gene. But as both of you said, the RNA template ma be defective. I wasn't sure what to make out of it when I repeatedly saw the PCR result but now I think it will be a good thing to start from a fresh harvest of RNA. The one I used for the template was just a presnt from the colleague.

T.

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