Dear All,
I have a ~2300 bp insert with Snabl and NotI respectively( 5 prime to 3 prime). I need to use pPIC9 expression vector which is 8 kb in size. I co digested pPIC9 with SnabI and NotI, gel purified and ligate with my insert. 50 ng of vector and 4:1 insert to vector molar ratio i used for the ligation. Initial incubation at 25 for 2 hours following over night incubation at 4 degree . I took 5 ul of ligation mixture after 48 hours ligation period, transformed in DH5aplha, mini preped and screened with Snabl and NotI. I got nothing. This is the third time I am doing. SnabI produce blunt end where Notl produces sticky end. Can you share your opinion? Am I doing anything wrong?
It may help to have internal controls- (i) I would run on agarose gel "ligated" DNA to check whether "ligation" reaction worked or not (basically disappearance of the insert on the gel); (ii) transform DH5alpha "competent" cells with empty vector alone. If restriction enzymes and ligase are from a good source (for example, New England Biolabs), it is likely that they are not contaminated with nuclease activity, thus, allowing ligation to proceed as expected. If the enzymes source is not reputed, it is hard to predict the problem.
You did mini preps, so you must have gotten some colonies. Do your colonies have only the vector without any insert? If so, you should treat the vetor with calf intestinal phosphatase to prevent self ligation. This will reduce the background with no inserts. You can also check your ligation by running a mini gel before and after ligation to see if your bands have altered migration.
What kind of ligase are you using? For T4 DNA ligase NEB recommends ligation at 16C overnight - not room temp. See their website for more tips. And are your restriction enzymes working? Try 2 small digests of your vector with each enzyme individually to make sure they're working. If one enzyme is bad the ligation is not going to work.
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