Load DNA PCR Product into Agarose Gel

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    • #17019
      9afa
      Participant

      Dear Scientist 🙂

      I am a new member 😀 and I have A question which I would like to share it with you to help me to get the reason.

      I am asking why do I got double band align under each other in Agarose Gel when I load DNA PCR Product ???
      Knowing that this DNA is Human DNA and I got it from Buccel swab and I am working in SNPs sequencing.

      Thanks in Advance.

    • #112929
      JackBean
      Participant

      What was your PCR DNA template? Did you check specificity of your PCR? Are you sure there is no splicing?

    • #112930
      9afa
      Participant

      Thanks a lot for your quick reply JackBean.

      Yes, I check all of what you said and It works at the first time without any problems, just suddenly it appeared without changing any things except I increased No. of cycling to 35 instead of 30 so is that a big deal and may appear as a double band in Agarose Gel ???

    • #112931
      JackBean
      Participant

      well, in such case, it could be that you had that band also before, it just wasn’t visible. The amplification was not that strong, but after 5 more circles the amount of DNA is 32-times higher.

    • #112932
      9afa
      Participant

      Ahaa, so do you think that will affect the sequencing??

    • #112934
      JackBean
      Participant

      depends what you want to sequence. Will you cut your amplicon and purify before sequencing?

    • #112943
      9afa
      Participant

      mmmm, I think yes because I did a PCR for particular reagion by adding a specific primer (exon) before sequencing .

    • #112947
      JackBean
      Participant

      but what is your teplate for sequencing? Is it plasmid DNA or the amplicon from PCR which have you purified from gel?

    • #112960
      9afa
      Participant

      amplicon

    • #112961
      JackBean
      Participant

      so you run it on gel, cut it from it and clean? Than it should be fine.

    • #112997
      9afa
      Participant

      Ok thanks 🙂

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