I am digesting my plasmid vector of size 6.6 kb size with Not1 enzyme (NEB). When I run it on the gel after digestion (after heat inactivating the enzyme at 65 degrees) I see the linearized single band, which I expect. But after doing phenol chloroform treatment and precipitating the linearized DNA overnight at -70 degrees, on gel I can see three bands. The most prominent band is even below 3 kb, faint band at the expected size and very very faint band above it. I do not know the reason why it is happening.
Thank you for the reply.
I have not done calf intestinal alkaline phosphatase. but if the vector is re-ligated also, still they should not run way below their expected size that is 6.6kb. The prominent band runs way below 3 kb also, around 1.5 kb. I have done this same vector digestion with same conditions earlier also but I never saw this problem.
Hi,
Self-ligation is possible and you should run on the gel along with your digested EtOH precipitated material the initial sample (intact plasmid DNA, which should be completely or partially supercoiled, that means, it runs faster than 6.6 kb). The result of self-ligation is a nicked pDNA, which will give you difference in mobility (thedifference will depend on whether you add EtBr into the gel or you "develop" your gel in EtBr solution after the run).
Another problem may be phenol, more precisely, its quality and, especially, pH.
Phenol should be colorless with pH around 8.0. Check the batch and buy a new one if required.
Good luck
Hi, If there is no problem with yield of DNA than go for gel/colum purification of DNA which will also get rid of contamination of phenol which hinders in down stream process. If you are getting three bands like undigested plasmid after precipitating is it possibility of self ligation. Instead of phenol pptn you can try simple Na-acetate pptn. Also check the color and pH of phenol, if there is a problem in phenol DNA will get degraded, and you will see smear of DNA on gel
I don't really understand the re-ligation part. How the phospho-diester bond will form by itself without ligase? Are the two enzymes you used identical? If not, there is even lesser chances of re-ligation even if ligase is present. I think changing the pheno can help.
Have you ever tried a (commercial) gel extraction kit. I have no idea where the smaller product comes from, my opinion would be an impurity in one of your reagents downstream of the gel electrophoresis, causing 'specific' breakdown.
The use of a gel extraction kit is much easier, and also important, more safe. There is no phenol to deal with.
Thanks, Syed - no ligase, no ligation. Even with phosphate.
Change your chemicals of circumvent phenol extraction.
Thanks everyone. I also thought of self ligation possibility. So i did redigestion of the religated DNA. It got digested and I saw only single band. Then I did alkaline phosphatase (CIP) treatment, followed by phenol chloroform treatment and then overnight precipitation. This time I have changed the phenol also. It is better this time because I can see prominent linearised band but still that religated band is there. Should I continue with in vitro transcription with the same linearized DNA. I am confused.
I am stuck with this problem since a week. Should i gel elute it? But the gel elution kit used in our lab gives every less yield.
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