Questions on failed T/A cloning

LOGAND logand at msdos.montpellier.inra.fr
Thu Sep 29 04:45:46 EST 1994


Dear All,

I have just attempted T/A cloning of a Taq PCR amplified fragment and have been 
unsuccesful. I am now wondering if this failure results from the fact that my 
oligo sequence is such that a nucleotide other than an 'a' has been added by the
Taq. So, while I intend to tootle off to the library and pick up copies of the 
articles cited on the net recently which will hopefully be able to tell me what 
base has been added, I have a couple of questions I hope can be answered by 
someone out there.

1. When a base is added to the PCR product by the action of Taq, is it always of
one type or is there simply a greater percentage of fragments that will have a 
certain base i.e. if another base is 'prefered' will I still have a chance of 
success, albeit with low efficiency, with a T-tailed vector ?


2. Can this problem be overcome by incubating my PCR product with Taq and dATP 
post-amplification, thus extending the product with the base I require ?

3. If 1 and 2 are not possible is complete blunt-end cloning, rather than trying
'g','c', or 'a' tailed vectors the way to proceed ?


Thanks in advance


David C. Logan
INRA-ENSA(M)-CNRS
Montpellier
France

Email: Logand at msod.inra.montpellier.fr  




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