Based on Fengzhang lab protocol provided on their lab website,I performed the one step digestion and ligation, I got a lot of colonies on negative control plate. I am wondering the cut sequence from the plasmid will be ligated to the plasmid, so why the negative control plate should no colony grows? The pX459 plasmid was used and I did not treat the ligation product with PlasmidSafe exonuclease.
Hi Hitoshi,
Thanks for your answer. If you digest plasmid first, you can treat the plasmid with phosphatase to avoid the problem. My question is how to explain the negative control result with single step digestion-ligation? Assume the plasmid was cut completely, the cut fragment still can re-ligated to the plasmid in one step protocol, so still can get colonies on negative-control plate.
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