Viewing 5 reply threads
  • Author
    Posts
    • #7654
      twomad
      Participant

      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.

    • #72744
      david23
      Participant

      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.

    • #72760
      twomad
      Participant

      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:

    • #72776
      david23
      Participant

      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.

    • #72796
      blcr11
      Participant

      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.

    • #72818
      twomad
      Participant

      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.

Viewing 5 reply threads
  • You must be logged in to reply to this topic.