Hello,
I'm trying to clone my amplified insert in pGEM-T vector using T4 DNA ligase and not getting positive clones even after trying variations in vector to insert ratio.
Amplification of insert was done using REDtaq Readymix PCR reaction mix.
Any suggestions would be appreciated.
The biggest issue with TA cloning is the loss of the T residue on the vector due to repeated freeze-thaws. You may need to obtain a new tube of TA vector if this has happened. Then aliquot the vector into the volume you would use for a ligation reaction (0.5 or 1ul) and freeze the aliquots. That way you are not freeze-thawing the entire amount of TA vector every time you need to do a ligation.
Does your DNA polymerase leave an A overhang? If not, buy the blunt-end kit OR using an A-tailing reaction to add on the required nucleotide.
Good luck!
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