ligation problem - self ligation of vector - (Mar/10/2005 )
perneseblue on Feb 24 2009, 12:35 PM said:
5 - T4 ligase and ligase buffer, both of these can and do go off rather easily. The T4 ligase enzyme in particular. Is anybody in the lab who is using this ligase suffering similar problems?
When you attempt to ligate your DNA again, I strongly suggest that you run a test. Run out some of the ligated DNA mix onto a gel (using a narrow well). If the ligation worked, you should see high molecular weight bands in addition to the bands that correspond to the vector and insert.
When you attempt to ligate your DNA again, I strongly suggest that you run a test. Run out some of the ligated DNA mix onto a gel (using a narrow well). If the ligation worked, you should see high molecular weight bands in addition to the bands that correspond to the vector and insert.
Continuing on...
You can test the ligase and the digestions quite simply. Treat some DNA ladder with ligase for ~20 minutes at RT, then run out on a gel. In a similar way take some of the insert and treat it. If the digestion has worked well, you will have a bit of monomer, as well as dimer, trimer and possibly higher -mers of the insert. If you only get a dimer-sized band, one of the enzymes has not worked properly. Similar with the DNA ladder; if the ligase is good, the ladder will shift upwards.
Some people have tried this test with the vector, both phosphorylated and dephosphorylated. The dephosphorylated version should remain monomeric, while the untreated should increase in size, but I think it's a bit of a dodgy expt, so I don't bother doing it. As others have intimated, I think you're usingfar too much AP for too long. I would follow the protocl exactly (it really can be that fussy an enzyme). Alternately, get hold of some Antarctic phosphatase; it seems more robust.
-swanny-