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PEG is the additon in so-called "quick ligase" buffer. It is very important in blunt end ligation, but tends to make more problems than it solves in cohesive end ligation (in my opinion). Also, reactions containing PEG cannot be heat-killed without dramatic loss of efficiency. I always do cohesive end, and avoid the quick ligase buffers.
#153328 Is ligation really necessary?
Posted
on 06 April 2013 - 08:21 AM
#153307 Is ligation really necessary?
Posted
on 05 April 2013 - 04:54 PM
Long matching overhangs can be directly transformed. Addition of PEG as a crowding agent is usually positive in making this work more efficiently. This does not work for overhangs of 4 bases.
#153163 Ligation troubleshooting! Please help!
Posted
on 31 March 2013 - 05:45 PM
Colony pcr with primers that bind only to the insert is a bad strategy. The insert DNA will still be present on the plate, and will amplify it.
You should use one primer on the plasmid backbone, and another on the insert.
Your ligation is likely prevented by the alkaline phosphatase. I would definitely avoid its use. You should not need it with a double digestion.
The colonies you see are likely undigested or singly digested plasmid which transforms. I would try again with no AP.
If you continue to have difficulty, I would suggest using PCR for amplifying the plasmid, which will dramatically reduce background.
You should use one primer on the plasmid backbone, and another on the insert.
Your ligation is likely prevented by the alkaline phosphatase. I would definitely avoid its use. You should not need it with a double digestion.
The colonies you see are likely undigested or singly digested plasmid which transforms. I would try again with no AP.
If you continue to have difficulty, I would suggest using PCR for amplifying the plasmid, which will dramatically reduce background.
