DNA gel to Test Ligation? - (Jun/25/2008 )
So, I've been trying to do some ligations followed by transformations and have been burning through competent cells and time, only to get colonies sometimes, and none at others. Is it possible to check the success of a ligation reaction on a DNA gel, or if only a small fraction of the DNA is ligated and won't show on a DNA gel? Thanks for your help.
It is a bit hard to test the actual reaction mix, because the concentration of the DNA is so low.
What you can do, however, is test the ligase indirectly. Take few ul of your DNA ladder and treat it with ligase for a few minutes, then run on a gel. If the ligase is working, you'll get a change in the ladder. You can alo use this technique to make sure your plasmid is double-digested. You should get a ladder of plasmid multimers: if your gel only shows a 2x size band, then one of your enzymes isn't working. Test with single enzyme digests of the plasmid to see which one isn't working.
If you really must know the success of your ligation, you could take 1ul of the reaction ad do a PCR using pasmid primers. This will also let you see how good the digestion was, because you product size and the mix of products will be different. The drawback here is that the PCR and gel will take a few hours, but if you do an overnight ligation reaction, you should still be able to complete the testing in one day and have time for the transformation if it has worked.
BTW, if you use a gel system without Tris, and only use borate, you can run a gel at 300 V for 10 minutes without excessive heating. Check out this Biotechniques paper: Rock and roll Gels! [attachment=4895:untra_fa...s__2004_.pdf]
For me it is a routine to check my ligation reaction by running an agarose gel. I can use half (5 ul) of my ligation reaction to run the gel and use the remaining for transformation. Controls (such as ligation reaction without insert, without ligase, etc) are necessary to judge the success of ligation.