how to confirm ligation is successful - (Mar/01/2006 )
Hi.Could anyone tell me what method can i use in order to know my insert and vector are successfully ligated?Can I use agarose gel electrophoresis to know if it is successful?I doubt it because if it is successful, the ligation product would be circular and I will not know whether the insert is ligated with the vector.
Well you will also monitor whether your insert band disappear from the gel
Then you can assume that the ligation was sucesfull but honestly I never do that test routinely you should just transform and make a pCR test that's the best !Pesji
i agree, transform and see if you get any colonies...then do PCR or minipreps....
I agree also, if its a complicated ligation and you expect to have few positive clones, then it is better to screen a lot of them. So better do PCR directly on the colonies.
If its simple ligation and therefore expect positives you may need the plasmid so better make minipreps. You can check by restriction analysis. Find enzymes that will give different restriction pattern if you have t he insert or not. Anyway, you may need to sequence your plasmid at the end, depending what you need it for, to check correct ligation sites, etc