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How to get double stranded cDNA from first-strand cDNA

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How to get double stranded cDNA from first-strand cDNA

Postby frankalpha » Mon Sep 20, 2010 8:08 pm

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.
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Postby BeyazRus » Mon Sep 20, 2010 8:23 pm

How long your cDNA strand is? Answer changes according to length.
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Postby JackBean » Mon Sep 20, 2010 9:20 pm

well, any polymerase would be fine having some primer
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: How to get double stranded cDNA from first-strand cDNA

Postby frankalpha » Tue Sep 21, 2010 1:52 pm

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


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

Postby frankalpha » Tue Sep 21, 2010 1:54 pm

JackBean wrote: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?
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Postby JackBean » Wed Sep 22, 2010 10:54 am

that depends on the polymerase used, but basically yes
http://www.biolib.cz/en/main/

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