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overlap pcr--who can help me - (Oct/07/2005 )

hi everyone,
Now i am doing overlap pcr to ligate two fragment ,one is 350bp,and the other is 2.2kb. The enzyme i used is Ex taq from takara. Butthe result showed that the non-specific band is clear ,while the band i want is in vague. Now i have tried several times, no success!

Your suggestions will be important to me .

-yanshi-

QUOTE (yanshi @ Oct 7 2005, 10:23 AM)
hi everyone,
Now i am doing overlap pcr to ligate two fragment ,one is 350bp,and the other is 2.2kb. The enzyme i used is Ex taq from takara. Butthe result showed that the non-specific band is clear ,while the band i want is in vague. Now i have tried several times, no success!

Your suggestions will be important to me .


In my opinion, annealing Tm is the most important factor. Maybe you could have a try by gradient PCR to find the optimal Tm.

Good luck

-Yang-