I want to ligate my 1kb insert (PCR product) with 6 kb vector, both are digested by ECORV (creating blunt end) and Sal1 (creating sticky end). How? Is blunt end may create problem in cloning?
In that case inserting fragment and vector can be ligated directly, blunt and sticky ends are incompatible as well as two different sticky ends. If vector completely digested and purified through gel-electrophoresis, it should be enough for succesful ligation.
Hi, I have recently done this same kind of ligation. my insert size was 2250 kb and vector 8 kb. I used 1:1 insert to molar ratio and incubated them at 16 C overnight. You can add 15% PEG 6000(2 ul) in a 20 ul reaction volume. I used SnabI and NotI
Hi,
I agree with Igor in terms of restriction end incompatybility.
In blunt end ligations the efficiency used to be lower than sticky end reactions. And there can be a problem that the orientation of the insert can be reverse. Self-ligation can be occur if the ends aren't dephosphorylated after cut.
The sticky end ligation can be more effective compare to blunt end. The insert orientation is straight. So you need to use the same restriction enzymes both insert and vector also for sticky and blunt end ligations respectivelly.
Bests,
Tamas
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