Subcloning large PCR products
DurochD at IRCM.UMontreal.CA
Sat Feb 3 14:55:21 EST 1996
>rosasg01 at mcrcr6.med.nyu.edu wrote:
>>For the last 3 months I have tryed to subclone a 8kb PCR product. So
>>far, nothing has worked. Does somebody has done something like this???
>>I'm sure somebody must have done it. If you have successfully
>>subcloned large PCR products, please let me know how big it was and
>>the protocol that you used for both PCR and subcloning. I'll really
>>appreciate any suggestions.
>>German Rosas-Acosta, graduate student
Maybe I can help,
I had similar problems regarding the cloning of a 6.3 kb PCR product. I
did my PCR using the eLONGase system (BRL). The polymerase was a mixture
of Taq and Pfu. Although Pfu has the exonuclease activity I was not sure
that all the PCR fragments had either blunt ends or had the dA extension.
Since neither blunt end cloning nor TA cloning had worked for me I taught
it might have been an "end problem".
After isolating my PCR fragment on gel and purified it using Qiaex II, I
did two things in parallel:
1) I polished my PCR fragment with T4 DNA polymerase and kinased the
fragment. I then dephosphorylated the vector (in that case pTz19R cut
smaI) and performed the ligation. It worked.
2) I incubated my PCR fragment with Taq polymerase for an hour at 72 C,
with only dATP as nucleotide. I was intending to do T/A cloning
afterward. I did not continue since I already had my clone but I do not
see why it should not work.
I hope it will help you,
More information about the Methods