TA cloning - (Mar/13/2007 )
I want to do the TA cloning of the expand long template PCR product. Is it possible to get the TA clones with the Roche amplified long pcr products ?
Hmmmm arent you using the non proofreading polymerase to create hanging A for TA cloning?
So you want to extend your PCR product? meaning both products with hanging A to clone to each other? Sorry.. I don't really get it.
Do you want to clone it into a cut vector with U or something else? 
So you want to extend your PCR product? meaning both products with hanging A to clone to each other? Sorry.. I don't really get it.
Do you want to clone it into a cut vector with U or something else?
I mean i have amplified the 6.4 kb gene by using the Roche expand template PCR system. Do you think TA cloning is possible with this PCR product?
It depends on the enzyme used in the Roche system -- what is it? Does whatever enzyme they use leave behind TA overhangs?
Yea... I agree with HomeBrew. Is it a non proof reading polymerase? or is it the proof reading one?
It depends on the polymerase. =)
It depends on the polymerase. =)
It contains Taq polymerase and TGO polymerase.
Should work, your amplified product contains a mix of -with overhang and -without overhang. We've succesfully TA-cloned with expand-hifi or expand-long template PCR products.
If you don't get enough colonies, add regular taq at the end of your reaction and have it another 10 minutes @ 72°C.