Restriction Endonuclease

[ecoli]

I'm running a restriction endonuclease reaction, with HIND III and BAM HI. These enyzymes should be cutting my PGEM vector, so I can add my desired gene in later. However, I'm trying to test whether or not the cut was successful by running the cut vectors on a gel. The problem, I keep getting two bands when I should be getting one. My prof. thinks this might be because the RE is not cutting properly. I haven't run a control yet, but I'd figure I'd ask here if anybody has encountered something like this before.

 

I'm not sure, if the RE is not cutting, where the two bands would come from. Anybody have any experiance with this and could spare a few ideas?

[Yggdrasil]

Sometimes when you run circular plasmid DNA, you will get two bands. The higher band corresponds to the "relaxed" plasmid and the lower band corresponds to the "supercoiled" plasmid.

 

However, the simplest explanation is that your RE digestion is incomplete. Some of the plasmid DNA is being cut, while some remains uncut. Since circular and linear plasmid DNA run at different positions on the gel, you would expect two bands in an incomplete digestion. Ciruclar DNA runs lower than linear DNA, so if you excise the higher band and gel purify that, you may be able to use that for your cloning steps. Perhaps you should try increasing your incubation time or the amount of enzyme used. Alternatively, you can try using a new stock of enzyme as your old enzymes may have gone bad.

[ecoli]

Thanks Tggdrasil, I'm not sure if my enzymes are degraded or not, how long do restriction enzymes usually hang around for?

 

My incubation time is ~24 hours, my assay calls for overnight, so I don't think that's the problem. I have a feeling that you're right about the enzymes being degraded though. I'll run a control to see if the enzymes aren't cutting or if its incomplete. Then maybe I'll get new enzymes, or do gel purification.

 

Thanks a lot for your help.

[Bluenoise]

Thanks Tggdrasil, I'm not sure if my enzymes are degraded or not, how long do restriction enzymes usually hang around for?

 

It depends on how they're stored. Repeated thawing and freeing can degrade them quickly.

 

If you can post a pic of your gel with the marker used and uncut plasmid size, it would be alot easier to give you a definite answer.

[ecoli]

It depends on how they're stored. Repeated thawing and freeing can degrade them quickly.

 

well, I'm not the only one in the lab to use them, so I dont really know how often they're used...

[newty]

Try cutting with only one enzyme. If the culprit is a bad enzyme, then you know your double digest is not working. Maybe the double digest reaction conditions are not optimal? Try a different buffer, more enzyme, let it go longer, etc.

 

Newty