Ligation problems... - (Oct/19/2008 )

I have no colony from my ligation. Here's what I did:

Digest insert/ UAST vector with NotI and EcoRI (each reaction is separate)
NotI 0.5ul
EcoRI 0.5ul
EcoRI buffer 2ul
BSA 0.2ul (used master mix to ensure more accurate pipetting)
DNA 2ul
water up to 20ul

Incubated at 37 deg cel for 1.5 hour, then 65 deg cel for 10 min.

Gel purified in 0.5% agarose. Bands were clearly defined for inserts, but still looked like a smear for the vector. Nonetheless, there is no unspecific band in the vector gel.



Next, ligate insert to vector
10X ligase buffer 2ul
UAST DNA 1ul (50ng total)
insert 1.5ul (1 insert:3 vector molar ratio)
ligase 1ul
water up to 20ul

Incubated at 16 deg cel overnight, then 65 deg cel for 10 min.



Next, transform 2ul into competent cells. My controls for transformation:
1) uncut vector
2) cut vector (with EcoRI and NotI)
3) cut insert (with EcoRI and NotI)

Uncut vector produced a lot of colonies, but the other controls and my ligation mixture produced none. So, controls work fine but for some reason, the ligation did not. Anyone has a clue?

The insert is typically at the higher molar ratio, but that is unlikely to be the problem. I'm concerned about your smear in the vector digestion. You could have problems due to UV exposure of the DNA during the gel isolation of bands. You can determine the ligation efficiency and quality of the insert and vector by ligating each independently, then dividing each ligation three ways. Cut one way with NotI, another with EcoRI. Run the NotI, EcoRI, and uncut ligations and check the ratio of single to double length fragments. Ideally, there should be only double length fragments. This will tell you whether the insert or vector, and which end, is causing problems.

How do you purify your DNA for ligation? High amounts of salt from the purification step could affect your ligation. Purification kits usually have a note explaining how avoid this problem.