Separation of double stranded DNA

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    • #14863
      roniadam
      Participant

      Hi everyone:
      I am interested to make a large insertion (780bp) in my vector. This 780 bp is a PCR product (megaprimer), I have used quikchange II mutagenesis kit but couldn’t success?? any idea about how to success???
      My thinking about the failing in this insertion is in the megaprimer ??? Any suggestion?

    • #104618
      JackBean
      Participant

      WHY do you have 780 bp primer? =-O

    • #104623
      roniadam
      Participant

      Hi Jackbean

      This is a piece of PCR product which is called megaprimer because I want to insert this piece into my vector.

    • #104624
      JackBean
      Participant

      and how do you want to insert it? By PCR or by digestion&ligation?

    • #104650
      roniadam
      Participant

      By mutagenesis kit (Quikchange II kit). It has been done before with another group and they succeeded in insertion but I was not. I emailed them but there was no reply that is why I posted this problem in this forum…. 🙁

    • #104663
      JackBean
      Participant

      OK, but how the kit works? By PCR, rigth? So why do you use such long megaprimer instead of using standard ~20mer?

    • #104668
      canalon
      Participant

      JackBean, I suggest you Google the system/kit before you make more of a fool of yourself. It does not work like a PCR.
      However I do not have a login to the Agilent website, and so could not explore the design of the primer. But that would be my best bet. That there is something going on with that specific primer. I imagine that the 780bp addition either do not anneal really well, or that it binds somewhere else in your plasmid. Maybe you can try conditions that are a bit more stringent (higher annealing temp, DMSO) if the kit allows that.
      Other solution, try megaprimer PCR out of the kit (create 2 PCR products that have 20-25 bp in common and use them as primers for 5 cycles then pause and add the primers that are at both end of the fusion and go on for 25-30 cycles. You should get a fusion gene that you can easily clone by restriction ligation.

    • #104670
      JackBean
      Participant

      I don’t know, what are you talking about, but I found this
      http://www.chem.uky.edu/courses/che554/quikchange.pdf

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