TA cloning

Patrick F.H. Lai pfhlai at LOOKSMART.COM
Wed Aug 19 19:00:47 EST 1998


---- Begin Original Message ----
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,

Barry Hudson
Dept. of Molecular Vascular Medicine
Level G
Martin Wing
Leeds General Infirmary
Leeds
LS1 3EX
0113 3923278
http://www.leeds.ac.uk/medicine/res_school/mol_vas_med/index.html
---- End Original Message ----

Dear Barry, 

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 >
Graduate Student
University of Toronto
Toronto, Ontario, Canada





       
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