I was wondering if anyone runs a ligation reaction on a gel to check efficiency of ligation before transformation? Basically, I took an aliquot of my ligation reaction and digested it with one restriction enzyme. Then I ran a gel. I didn't get any bands. Some people say you can't run a ligation reaction on a gel because the small amount of DNA won't show up. Does anyone have an opinion or experience with this?
Though I have not done I heard people doing gel run to check for ligation success before transformation. But as I said I never did, and my ligations always worked with no troubles. Right proportions of vector and insert and good quality ligase and buffer is what you need for success in ligation. 3 hrs at 22degreesC is enough and if you are using quick ligases hardly minutes before you transform.
see the ligation product on a gel will depend on the amount of DNA you sow. I recommend you to do a colony PCR with primers that annealing on the vector to see if you got linked product.
You need about 20 ng of DNA to clearly see a single band on a gel. So I imagine you ligated about 100 ng in total of vector plus insert. Then you took an aliquot for digestion... maybe 2 uL from a 10 uL reaction. Assuming that you loaded the entire digest on the gel that would mean you loaded about 20 ng of DNA which would yield a faint band. However, I assume you digested the DNA to look for a restriction pattern containing more than one band. Hence the amount of DNA per individual band would be less than 20 ng.
I would normally try to load about 50 ng of uncut ligation onto the gel so I could see it easier. Ligated product will give you higher molecular weight bands than if you just loaded the two fragments pre-ligation. You can tell if your DNA has ligated if you load the pre-ligated DNA in the next lane for comparison.
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