Wednesday, 4 April 2007

cloning - Deletion errors with Phusion Polymerase?

In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen.



I have seen deletions with T4 DNA polymerase during "blunting" reactions. According to the NEB website:



Q10: Is T4 DNA Polymerase active at room temperature?



A10: We suggest 12°C. The DNA ends "breathe" at higher temperatures allowing the exonuclease to remove nucleotides past blunt.

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