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    • #13058
      jasmina
      Participant

      hi there!
      I want to know how to get a aim band size using BLAST, when 2 primer bands and aim gene name are given.
      is there anyone who can help me? thank you O(∩_∩)O~

    • #98819
      JackBean
      Participant

      just download the sequence of your gene and look, where the primers anneal and from that calculate the size of your amplicon.
      E.g. if your fw primer anneals to +25 nt and rev primer anneal to +450, than your amplicon will have 450-25 = ~425 nt

    • #98821
      jasmina
      Participant
      quote JackBean:

      just download the sequence of your gene and look, where the primers anneal and from that calculate the size of your amplicon.
      E.g. if your fw primer anneals to +25 nt and rev primer anneal to +450, than your amplicon will have 450-25 = ~425 nt

      but in most cases, primers are small, sometimes they can anneals to several site, what should i do in this case? 😕

    • #98825
      JackBean
      Participant

      the primers should be long enough to be quite unique at least for your species.
      At PubMed’s BLAST is some tool for designing primers
      http://www.ncbi.nlm.nih.gov/tools/prime … =BlastHome
      but I have never used it. Anyway, if you already have your primers, just use BLAST to see, where can it anneal.
      Also, as you use two primers, you highly decrease the probability of inspecific amplification

    • #98833
      MrMistery
      Participant

      if your primers anneal to multiple places, then that’s a problem in itself.

    • #98836
      jasmina
      Participant

      Thank you ! O(∩_∩)O~ i get it. first download aim gene mRNA sequence ( select genus) ,then blast 2 primers and mRNA sequence with blastn-suite respectively, after get annealing site, calculate band size, right?

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