TA cloning

[malbrech at asmoday-atm.rz.uni-frankfurt.de]

Patrick F.H. Lai (pfhlai at LOOKSMART.COM) wrote:
> Dear Patrick,

> Thank you for your reply to my query.  I was wondering if you could give 
> me a few more details about how you A and T tail your products.  I have 
> tried this way of cloning blunt products by:
> 1.  Assuming Taq adds on an extra A tail in the PCR reaction and using a 
> cycling profile of:  940C for 5 minutes to denature,  and then 30 cycles 
> of 940C for 1 min, 60 0C for 1 minute, 72 0C for one minute.  This is 
> followed by an extension step of 5 minutes at 72 0C and stopped at 4 0 C.
> 2.  T-tailing SmaI digested pGEM-3Zf at 72 0C for 10 minutes followed by 
> phosphatasing the plasmid.

> Thanks again,

Dear Barry

Patrick wrote in his message

> But I would suggest lengthening the extension step to at least 10 min
> at 72 degC to make sure the Taq polymerase would unspecifically tag 
> on the extra A at the ends of as many strands of DNA as possible.

and also in my hands this allways worked perfectly.
 
> About the vector, I honestly have not done this with success.
> I tried once, it didn't work, and my generous boss told me to buy the 
> TA-cloning kits.  I have used pGEM-T Easy, pCR-II and pCR2.1-TOPO.
> All worked beautifully.

Yes these vectors are quite good but they cost a lot and when I was
doing this kind of cloning I read the following paper:
Borovkov, A.Y.,Rivkin,M.I.  BioTechniques 22,812-814 (1997): XcmI-
containing vector for direct cloning of PCR products.
When I contacted the author he sent to me the plasmid which is a 
pBluescript derivative (blue-white-screening) 
and it worked perfect for all subsequent TA-clonings we did in our group.
Hope this helps.

Manuela Albrecht
Bot. Institut
JW Goethe Universitaet Frankfurt
e-mail: M.Albrecht at em.uni-frankfurt.de