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amplification of 14Kb - (Jul/16/2012 )

Hello All,

I need help from you to amplify a 14kb insert.
I have been trying to amplify this using TAKARA LA Taq(Long amplification taq) with different optimization steps of annealing temperature,cycles,Mg+ and so on.
but i could get only a smear I also tried with different polymerases but in vain

Is there any other possible way to amplify.
like breaking this whole 14kb into 2 or 3 parts ,amplify separately and later join them and then use them finally for transformation?

Any suggestions will be happily received.

Thanx in advance.



Is your target gene GC rich? IF yes, you might consider add DMSO, BSA or Betaine as addictives.
Also, can you describe your PCR conditions and mastermix in detail? It will be very hard for us to troubleshoot for you without sufficient information.

-Adrian K-