How to get double stranded cDNA from first-strand cDNA

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    • #13795
      frankalpha
      Participant

      As the title goes, if I want to get double stranded cDNA from the single stranded first-strand cDNA without conducting a PCR, What should I do?
      According to this link ‘http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm’, seems I can use DNA Polymerase I to do it, using the hairpin loop as the primer or using the RNA leftovers as primers after RNase digestion. However, wikipedia says DNA Polymerase I is insufficiently processive to copy an entire genome, as it falls off after incorporating only 25-50 nucleotides. Looks like this enzyme is not very efficient and active. Any alternatives? Or maybe just use any commercial Taq polymerase?
      Thanks.

    • #101356
      BeyazRus
      Participant

      How long your cDNA strand is? Answer changes according to length.

    • #101362
      JackBean
      Participant

      well, any polymerase would be fine having some primer

    • #101390
      frankalpha
      Participant
      quote BeyazRus:

      How long your cDNA strand is? Answer changes according to length.

      The strands are 10 kb and 6 kb respectively. Thanks.

    • #101391
      frankalpha
      Participant
      quote JackBean:

      well, any polymerase would be fine having some primer

      How’s the reaction condition? Just add the polymerase and use the assumed primer and set probably 20 PCR cycles, i.e. denaturation – annealing – extension?

    • #101415
      JackBean
      Participant

      that depends on the polymerase used, but basically yes

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