Login

|
|
How to get double stranded cDNA from first-strand cDNAModerator: BioTeam
6 posts • Page 1 of 1
How to get double stranded cDNA from first-strand cDNAAs 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.
Re: How to get double stranded cDNA from first-strand cDNA
The strands are 10 kb and 6 kb respectively. Thanks.
Re:
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?
6 posts • Page 1 of 1
Who is onlineUsers browsing this forum: No registered users and 1 guest |
© Biology-Online.org. All Rights Reserved. Register | Login | About Us | Contact Us | Link to Us | Disclaimer & Privacy