Subcloning large PCR products

[Daniel]

>rosasg01 at mcrcr6.med.nyu.edu wrote:
>
>>hElP!!!
>
>>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.
>>Thanks.
>>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,

Daniel