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False positive for PCR screening of insert - (Jan/17/2006 )

Not so long ago this happened to me: I've cloned my desired gene into a cloning vector and transformed it to E. coli. I got colonies that I've screened with PCR for positive clones. I got some and made a plasmid prep out of one and then checked with PCR again, all good, fragment is there. BUT, when I went on with sequencing the region, I found out that my insert wasn't there and the whole sequenced bit was completely complementary to the multiple cloning site of my vector.

The mystery is: how can a PCR check-up show that the insert is there when it's not!?

My PI told me that I shouldn't trust PCR always and instead do digestions, and that did solve the thing, but this "phenomena" still puzzles me.

What do you think?

-smoochiepie79-

The only explanation is contamination. There is no other mysteries.

-pcrman-