A-tailing with Taq

[Patrick F.H. Lai]

---- Begin Original Message ----
>   I have always read that Taq has a preference for adding an extra >"=
A" at the end of a PCR product, however, does anyone know >semi-quantita=
tively
>(for example, a number between 25% and 100%) how often this happens? >=
I was also wondering if I should add some extra dATP at the end of my >P=
CR reaction, and let it tail for 10' at 72C, rather than just >letting t=
he reaction "tail" for 10' at 72C at the end of the PCR.
>   Any suggestions?
>
>Thanks,
>Koichi Kunitake
><kunitake at salk.edu>
>
---- End Original Message ----


Dear Dr. Koichi Kunitake,

For the purpose of TA-cloning into pGEM-T Easy, I never had to do 
extra work.  The 10 min of 72 deg C incubation after 25~35 cycles 
was sufficient for my cloning purposes.
I tried Taq polymerase from BM and Pharmacia, and both worked for me.
However, if your DNA fragment is really AT-rich, adding extra dATP 
does not sound like a bad idea.

Don't worry, my fellow researcher.
TA cloning works, especially when you are using kits.
As long as you put enough inserts in your ligation reaction,
I am sure you will get more big, white colonies than you want on your 
agar plates.  
(However, if you are T-tailing your own vector, 
then it may be another story.)

Good luck.    :-)





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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