I have a PCR product that I've been using as a component that I'm ligating into a vector. Did sequencing it was confirmed to be correct and did a ligation it worked perfect and was sequence verified. I did two more ligations using the same PCR product into different vectors and now there is a mutation in the PCR product? How does this happen? I've essentially sequenced it twice(one time just the PCR product and then again to make sure the it was ligated correctly) and it was fine and now the sequencing is showing a deletion. I send to two different companies for sequencing and they have both come back with the same results each time. The first 2 times both showed it to be correct the last 2 times there is a mutation that both companies are picking up on.
When you do PCR amplification, the quality of the polymerase is important, as it can introduce mutations. High-fidelity polymerase are available and must be used.
Pick a few more colonies and see if they have the correct sequence. Mutations happen all the time at low levels, it's normal to need to screen a few possible positive colonies to get one that has the correct vector sequence.
Good luck!
Sounds like you maybe had a mixed PCR product pool, some were fine and others had the mutation. You could try sub-cloning your PCR product (check the sequence in the sub-cloning vector) then using cut and paste to move it from there to your final vector. It does take time but can reduce the odds of a mutation in your final clone.
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