Patrick F.H. Lai
pfhlai at LOOKSMART.COM
Wed Aug 19 19:00:47 EST 1998
---- Begin Original Message ----
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.
Dept. of Molecular Vascular Medicine
Leeds General Infirmary
---- End Original Message ----
I am not that experienced in this, actually.
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.
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.
You may want to search the archive here at net.bio.net, and also the
FAQ section. There are quite a bit of useful stuffs on this trick.
Hope this is useful info. :-)
Patrick F.H. Lai < PFHLai at looksmart.com >
University of Toronto
Toronto, Ontario, Canada
or keep looking.
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