Plasmid Digestion

[eternalmetal]

I am running a restriction digest with the Pyp04 plasmid (has 1 restriction site for BglII) and BglII. Using 2ng of plasmid, I was told to use 5 uL of BglII to digest. After 2 hours of digestion I ran a gel to test how well my sample was digested, but did not include an undigested sample (I am under the assumption you wont just see one band if it was under digested). The bands were a little on the light side, so I am doing an etbr soak to brighten the gel up. In the meantime (1.5 hours after I started the gel), I was told to add another uL of enzyme to the digestion for another hour. So my question is, would it really be necessary to do so? A typical digestion of this amount would be done with less enzyme and would most likely be fully digested in an hour's time.

 

Im kind of new at this so im just looking for a "what would you do" type of answer. Imo adding an extra uL of enzyme would be unnecessary based on protocols so I didnt add it.

 

Opinions? thanks in advance

[ecoli]

You could always try doing it both ways and comparing the samples side by side

[CharonY]

The way to assess this is to know how much enzyme (in terms of activity or units, not volume) compared to the DNA. If what you describe is correct and assuming that the enzyme is not diluted and the right buffer is used you are doing a horrible overdigest anyway.

Low concentrated enzymes are mostly sold with around 10 units/ml. 10 units (i.e. 1µl) is generally sufficient to digest around 2 µg of DNA within an hour under optimum conditions (depending on DNA it may even take less).

You are using potentially 50 units for 2 ng. However, even if it was a typo and you used 2 µg (as 2 ng diluted in buffer would operate below the detection limit of EtBr gels) it should be more than enough. Especially as BglII had star activity, if memory serves.

 

But more importantly, what is the rationale behind adding more enzyme? Low intensity in gels would not be a good reason.

[eternalmetal]

The way to assess this is to know how much enzyme (in terms of activity or units, not volume) compared to the DNA. If what you describe is correct and assuming that the enzyme is not diluted and the right buffer is used you are doing a horrible overdigest anyway.

Low concentrated enzymes are mostly sold with around 10 units/ml. 10 units (i.e. 1µl) is generally sufficient to digest around 2 µg of DNA within an hour under optimum conditions (depending on DNA it may even take less).

You are using potentially 50 units for 2 ng. However, even if it was a typo and you used 2 µg (as 2 ng diluted in buffer would operate below the detection limit of EtBr gels) it should be more than enough. Especially as BglII had star activity, if memory serves.

 

But more importantly, what is the rationale behind adding more enzyme? Low intensity in gels would not be a good reason.

 

I used the correct buffer without diluted enzyme, and yea I meant 2 ug

 

The rationale between using more enzyme was that an experiment someone else did 6 months ago did not turn out, and who I report to thinks that the underdigestion of the plasmid was the culprit. The goal is to open up the plasmid in order to add an insert and transform into e. coli. Im no expert, but I dont think that this type of application would require extra digestion as compared to your typical experiment. Or does it?

 

I just dont want to learn by making a mistake that I was led into making. If it were up to me I would have used 2 uL of enzyme.

 

Thanks for the reply Charon

[CharonY]

As mentioned above 10 units of restriction enzymes should be sufficient to completely digest 2 µg DNA in one hour. The only reason to add more enzyme is when the enzyme lost activity. Otherwise it won't do anything and in case of star activity it would be harmful.

[jimmydasaint]

You can do a check of your DNA to see how 'clean' it is from protein before the digest. You may have to double extract it (using chloroform/ether IIRC) before digestion. I'm not sure but I just wonder if it may be worth an SDS PAGE of the enzyme to see if you get a smear on the protein gel, or well defined bands? Staining techniques have a high level of sensitivity, including Coomassie Blue with a sensitivity of 5-25 ng per band if the figures are credible. Stain

[CharonY]

Actually just using the 280/260 ratio would be much faster. However I do not really see how that relates to the OP?

[jimmydasaint]

Actually just using the 280/260 ratio would be much faster. However I do not really see how that relates to the OP?

 

Sorry Charon Y, I have to learn joined up thinking sometime. I was considering the possibilities that:

 

a) the DNA was not clean enough for digestion and a double extraction could help;

 

b ) the restriction enzyme could have degraded or been contaminated and an SDS PAGE would help to show its purity, although there are quicker methods.

 

 

Just thinking back 20 years or more makes my head hurt so I'll leave it to people who still do proper experiments.

 

 

SDS PAGE would be unlikely to help for higher molecular weight enzymes anyway IIRC.

Edited July 17, 2010 by jimmydasaint

[CharonY]

Ah, I see your point now. Of course it is only speculation, but based on what is described it does not appear that there is any indication of underdigestion. If it was one would easily see the undigested plasmid. Unfortunately SDS is only really useful if you have got a massive degradation of the enyzme (and even then degradation products are often not easy to see, depending on how the gel runs). It would have made sense if the enzyme was purified by oneself, but nowadays I hardly think anyone is doing that anymore. Well, outside of classes that is.

[eternalmetal]

What would you clearly see if the plasmid was underdigested?

 

Im assuming if it was digested too much it would look like this:

 

I didnt see any extra bands, but I guess if I wanted to verify if it was digested I would have just run an undigested sample side by side for comparison. There werent any problems with the experiment (as far as I know so far), this topic was purely speculative so that I could understand more about enzyme digestion.

 

Thanks again for all the help