T4 ligase buffer - (May/20/2010 )

Ok, so I have another odd one. I have DNA which was double digested and gel extracted that I am using for a ligation. My ligations have not worked so I decided to see where things are going wrong. I ran a gel with 3 variations of the DNA: 1) directly thawed from the freezer, 2) mixed vector & insert in a tube and set on bench (to mimic ligation conditions), and 3) mixed vector & insert with ligase buffer and set on the bench. After running this on a gel I am more confused.... 1 & 2 both show the correct sized bands for the digested vector and insert, tube 3 shows a band of higher M.W. than is expected.

....bueller? bueller?

Any ideas?

1. Circular plasmid does not migrate as linear DNA - it migrates usually slower
2. PEG or some other component in ligation buffer (DTT?) do not work well with gel electrophoresis and thwarts correct migration - my experience

If tube 3 shows only one band, then you have nothing to worry about.

You could also run a fourth lane with everything but the plasmid and insert as a secondary negative control to make sure that the slower band you saw on the gel is the ligated product you expected.

georgiadave on May 21 2010, 05:06 AM said:

Am I being dim, or do you not have any tubes with vector, insert, buffer AND ENZYME? Surely, that is the actual ligation reaction... :-)

If you're not sure if the ligase is working properly, set up a couple of diagnostic reactions;1) DNA ladder as the ligating DNA; 2) insert as the ligating DNA; and digested vector as the ligating DNA. Incubate at RT for 20 min or so and run on a gel, along with unligated ladder. The ladder should shift up, the insert should start to make a ladder, as should the vector DNA, but it might be hard to see this. If the insert doesn't make a ladder, then one or both of your enzymes, if you're using two for directional cloning, isn't working; if it makes a dimer-sized band, then one enzyme isn't working. If the DNA ladder doesn't shift, then the ligase or perhaps the buffer is off.

Oh, yes, the ladder can't have loading dye in it.

Swanny, thanks for the outline!

You are not being dim, i did not include a lane with ligase. I thought to look at things this way because I suspected DNases to be in my sample that were being activated by the Mg in the ligase buffer. The fact that there was intact DNA present was surprising - shifted DNA more surprising. I am simply curious if others have experienced this and what they found to be the cause.