Issues with ligation (View forum version)



ASNP

Posted 29 November 2011 - 04:25 AM

I have been trying to clone a 350bp fragment in pTZ57RT for the past few weeks. TA cloning that is supposed to be so simple is giving me a lot of problem. Transformation is fine (as my controls work) but the ligation never works. Can anybody suggest me the optimum conditions for my ligation reaction?

I usually set up a 15uL reaction with vector: PCR prdt ratio 1: 3 and incubate overnight at 4C.

My PCR product is 350bp long, TA cloning kit we use is Fermentas InsTA clone kit and the cells I use for transformation are E. coli DH5a.

Thanks in advance.

phage434

Posted 29 November 2011 - 05:25 AM

Are you using an enzyme that leaves A tails?

Are your cells really competent, or are they only sort-of competent? (Three or more orders of magnitude difference).

mahrak

Posted 08 January 2012 - 07:31 AM

i suggest you treat your TAcloning vector with dATP as they tend to lose the terminal dATP as time passes.