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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 ?

-sktewary-

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? wink.gif

-timjim-

QUOTE (timjim @ Mar 13 2007, 11:01 PM)
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? wink.gif



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?

-sktewary-

It depends on the enzyme used in the Roche system -- what is it? Does whatever enzyme they use leave behind TA overhangs?

-HomeBrew-

Yea... I agree with HomeBrew. Is it a non proof reading polymerase? or is it the proof reading one?
It depends on the polymerase. =)

-timjim-

QUOTE (timjim @ Mar 14 2007, 08:21 PM)
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 contains Taq polymerase and TGO polymerase.

-sktewary-

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.

-vairus-