How to get double stranded cDNA from first-strand cDNA

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
frankalpha
Garter
Garter
Posts: 9
Joined: Mon Sep 20, 2010 3:36 pm

How to get double stranded cDNA from first-strand cDNA

Post by 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.

BeyazRus
Garter
Garter
Posts: 12
Joined: Sun Sep 19, 2010 8:07 pm

Post by BeyazRus » Mon Sep 20, 2010 8:23 pm

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

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by 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.

frankalpha
Garter
Garter
Posts: 9
Joined: Mon Sep 20, 2010 3:36 pm

Re: How to get double stranded cDNA from first-strand cDNA

Post by 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.

frankalpha
Garter
Garter
Posts: 9
Joined: Mon Sep 20, 2010 3:36 pm

Re:

Post by 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?

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Wed Sep 22, 2010 10:54 am

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

Cis or trans? That's what matters.

Post Reply

Who is online

Users browsing this forum: No registered users and 4 guests