TA ligation - molecular ecology

[Andrew O'Neill]

I am experiencing difficulty in ligating purified PCR amplified, mixed community
16SrDNA from a sediment sample.  The kit is manufactured by Promega and all 
controls, including ligation control are working according to specs.  However, my 
sample does not ligate into the pGEM-T vector.  Any tips, experience with this 
problem? I have also tried excising the 1.5kb band from an agarose gel and 
purifying, and an A-tailing reaction post-PCR.  

I would really appreciate any advice sent to my mailing address, as e-mail access is 
more readily available than internet.

Thanks in advance,

Andrew O'Neill 

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Andrew O'Neill B.Sc. (Hons) PhD student
Centre for Bacterial Diversity and Identification
University of Qld, St Lucia, Qld, 4067
email: oneill at florey.biosci.uq.edu.au
ph: +61-7-3365-3211
fax: +61-7-3365-1566
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