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Overlap PCR failing to introduce mutations

Overlap PCR failing to introduce mutations - Molecular Biology Techniques

Overlap PCR failing to introduce mutations - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 02-28-2010, 09:12 PM
Pipette Filler
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Question Overlap PCR failing to introduce mutations



Hi,

I attempted to use Overlap PCR to introduce two mutations (F to A mutation) into the DNA sequence of a protein im studying. The mutations were carried on two seperate primers i.e. one on primer B and the second on primer C. PCR products AB and CD were the correct size when checked on a gel. Again the final product was the expected size.
However after sending the final PCR product away for sequencing, the results i recieved back indicated that no mutations had been introduced and after a blast was proformed it was a 100% identical to the wild-type proteins DNA sequence.
Can anyone help me understand why that happened? and what i can do troubleshoot this?
would appreciate it
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Old 03-02-2010, 09:50 AM
Pipette Filler
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Default Re: Overlap PCR failing to introduce mutations

I can't really help you with troubleshooting, but I recommend trying the "QuikChange Site-Directed Mutagenesis Kit" by Stratagene. It has never failed me!
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failing , help with overlap pcr , introduce , mutations , no mutations via pcr , overlap , overlap pcr , pcr


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