I have double digested and gel-purified pET22b vector and put ligation with my insert in 1:3 & 1:5 and kept at room temperature and 16C overnight with both, transformed into DH5a, plated them into LB Ampicillin plates (100 ug/ml, 200ug/ml and 300 ug/ml). But all the plates are giving lawn. After doing PCR, there was no band for the insert.
Kindly help.
You write that all the plates are giving a lawn, if this is true then either your ampicillin plates have gone bad or else your host strain is already amp resistant. Yes sure to always do your transformation controls of undigested plasmid to ensure you get lots of colonies (should be tens of thousands) and no plasmid control which should give a clean plate.
Another option: Not easy to verify double cuts, so I always come from a backbone, which has already a working insert.In my case it is a GFP gene in all the vectors I us.I always use antarctic phosphate like suggested already.
Also nice option: Digest the vector eg with additional PstI (in the amp gene).Then perform a 3 fragment ligation, which should work always and impossible to get background.NdeI could be troublesome due to the short overhang and giving high backgrounds, if the double cut is not verified.So cleave eg NdeI/PstI, PstI/NotI for example , isolate the fragments and include your fragment then in the ligation: eg NdeI/NotI fragment.Should give no background.Always include a control fragment as test, if you have your source plasmid with an insert already.
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