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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?


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