TA cloning question
Posted 03 December 2004 - 01:02 PM
I am really upset for I try to ligate the insert 400bp to 4k vector but it keeps failing. The problem may be the poor cleavege of insert. So I plan to try TA cloning to increase the cleavege efficiency of the insert.
For TA cloning, the A nucleotide is required at 3'. I know a lot of Taq polymerase will automatic add A at the 3' but I don't know the iTaq polymerase from Bio-rad has the same function. I really don't wanna start over the PCR again.
Could any experienced friends give me some help? Really appreciated!
Posted 04 December 2004 - 02:20 PM
My best suggestions are first, connect to BioRad's technical support. I am pretty sure that you can clone your product without extra procedures. Second, if you are using TA, overnight ligation is preferred.
My best wishes of luck for U!
Posted 04 December 2004 - 04:54 PM
I believe that iTaq can add 'A' to PCR product, better ask Bio-Rad.
If you have your PCR product ready, although fresh PCR products are recommended for TA cloning, you can still use the PCR product for ligation. Before ligation, cycle your old PCR reactions (not old than one week) 1-2 times and extend at 72C for 5 min. Then purify the reactions and do the ligation.
5 min ligation also works well for some systems.