Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/04/09
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Ryan proposed that during the heat inactivation stage of the EX double digestion (for the synthases), the polymerase from the PCR reaction may have been activated again. This would allow the polymerase to fill in the overhangs created from restriction enzymes, which would be a valid reason for why the inserts aren't inserting correctly into the backbone.
So, Ryan treated the PCR products of the synthase genes with 5U of alkaline phosphatase for 3 hours and then heat inactivated. This removes the 5' phosphates which will prevent the polymerase from filling in future sticky ends that we create. I double digested these treated synthases, did not heat inactivate, and then PCR-purified them. Concentrations ranged from 4ng/uL–33ng/uL.
Next step is to gel-purify the backbone, and then we can try the ligation again.