T4 ligase buffer
Posted 20 May 2010 - 11:06 AM
Posted 20 May 2010 - 11:27 AM
2. PEG or some other component in ligation buffer (DTT?) do not work well with gel electrophoresis and thwarts correct migration - my experience
Posted 20 May 2010 - 03:13 PM
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.
Posted 20 May 2010 - 08:50 PM
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.
Posted 21 May 2010 - 08:02 AM
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.