Hi Mahesh,

There could be few reasons.  Firstly, there may be contaminating nucleases, either in the tube which contained your double digest, or in the NotI enzyme / reagents.  It looks like your uncut plasmid is ok, so your plasmid DNA isn't degraded.  I'm not sure how long you performed the digest for, but if it was an overnight digest, it might be that the enzyme overdigested the product.  Try to replace your water, use a different buffer, use new tubes and then digest your plasmid for an hour and run on a gel and see if this helps.  If this still results in a smear, you may want to look into using another aliquot of NotI. 

Hi Mahesh,

There could be few reasons.  Firstly, there may be contaminating nucleases, either in the tube which contained your double digest, or in the NotI enzyme / reagents.  It looks like your uncut plasmid is ok, so your plasmid DNA isn't degraded.  I'm not sure how long you performed the digest for, but if it was an overnight digest, it might be that the enzyme overdigested the product.  Try to replace your water, use a different buffer, use new tubes and then digest your plasmid for an hour and run on a gel and see if this helps.  If this still results in a smear, you may want to look into using another aliquot of NotI. 

Dear Mahesh,

Would agree with Juliette on setting up the double digest again making sure everything you use is nuclease free (especially the tubes and water). Type 2 restriction endonucleases like NotI should not over digest since they are very specific. I have always set up overnight digests for that matter (even with high fidelity enzymes).

Abhishek

a single digest with not1 would be useful. If That gives a smear then the Not1 is definitely contaminated. It is commonly used so perhaps you can borrow some Not1 and buffer from another researcher

I agree with Paul, because i had the same problems, the enzyme may be contaminated or the buffer may have problems as well. Do single digestion and then see.

Did you incubate the uncut sample in same buffer and for the same time? That will tell you if the problem comes from enzymes or buffer or from the DNA prep

I would recommend you to set up a new experiment with shorter time of digestion, fresh buffer and fresh batch of Not1 enzyme. There should be no contamination in the reaction at any phase.

Dear Mr. Mahesh, 

I am agreed with Juliette Delhove, kindly follow the instructions given by her.

Best of luck