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semi-random PCR questions

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#1 TracyDuke



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Posted 21 January 2011 - 03:06 PM

I designed an experiment to identify chromosomal translocation involving gene of interest.
Since the fusion gene contain my gene of interst (X) and an unknown partner gene, I plan to use a PCR+cloning+nested PCR strategy:

1)PCR using one primer hybridizing gene X and a combination of 3 short random primers. PCR is carried out at a low anealing temperature (42 degree)

2)The PCR products are then cloned into a T-A vector, transformed and individual clones are grown to get plasmid DNA. I expect a lot of clones are just non-specific, meaning that they do not even contain any part of gene X.

3)Do a second round PCR using an nested (inner) primer specific to gne X in combination with either T7 or SP6 promoter primer, which hybridize the T-A vector itself. Therefore,the clones do not contain gene X will not be amplified. And I expect that the PCR product should contain at least a portion of gene X.

4)PCR products from step 3 are cloned into T-A vector again and sequenced to identify fusion genes.

Does this stratagy sound OK? My currunt problem is that the majority of the clones from step 4)have nothing to do with my gene of interest. Any comments are greatly appreciated. Or drop me a message if you are interested in knowing more details. Thanks a lot!

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