Problems with certain restriction enzymes - on the verge of tears - (Sep/21/2007 )

Hi all,

I hope you can help. My work has been held up for about three months now and I am tearing my hair out.

I am having problems with certain restrition enzymes and when I digest vectors (it only seems to happen with vector digestion). I am getting a smear (see picture - lane 1 is marker, lane 2 is BamHI, lane 3 is HindIII, - ignore the other lanes). I cannot tell if it is star activity or DNase contamination. I am thinking it is star activity as only a few enzymes are affected namely HindIII, SacI, PstI.

I have tried everything including changing vector preps (my prep is Qiagen mini-prep and v pure), changing enzyme supplier, changing tubes, autoclaving tubes/tips, changing the amount of enzyme used, changing the incubation time. You name it.

Has anyone seen this before? Is it star activity?

If anyone has any suggestions then it would be a great help.

thanks v much

Ooops no picture. Here it is



Hi all,

I hope you can help. My work has been held up for about three months now and I am tearing my hair out.

I am having problems with certain restrition enzymes and when I digest vectors (it only seems to happen with vector digestion). I am getting a smear (see picture - lane 1 is marker, lane 2 is BamHI, lane 3 is HindIII, - ignore the other lanes). I cannot tell if it is star activity or DNase contamination. I am thinking it is star activity as only a few enzymes are affected namely HindIII, SacI, PstI.

I have tried everything including changing vector preps (my prep is Qiagen mini-prep and v pure), changing enzyme supplier, changing tubes, autoclaving tubes/tips, changing the amount of enzyme used, changing the incubation time. You name it.

Has anyone seen this before? Is it star activity?

If anyone has any suggestions then it would be a great help.

thanks v much
[/quote]

have you changed digestion buffer? Could the buffers be contaminated?

Can you list your digestion mix? Perhaps we can spot something there. Star activity for HindIII, PstI and BamHI is rare if the enzymes and the correct buffers used at the right concentrations are use.

I don't think it's star activity because the BamHI band is larger than what you see with the other enzymes, and star activity would produce smaller bands. Also, I think you are loading too much marker, because the bands are far too 'smiley', especially at the top of the gel. If you're having trouble seeing things, stain for longer.

Step 1 would be to repeat the experiment with someone else's enzymes. Next, I would suggest you re-precipitate your plasmid again.

What does the undigested plasmid look like on a gel? For t hat matter, how do you purify the plasmids in the first place?

Hello,

I have use Promega enzyme and Invitrogen enzymes in the recommended buffer. My reactions have contained

2ug plasmid in 15ul H20
1ul enzyme (10u/ul)
2ul x 10 buffer (as recommended and supplied by manufacturer)
2ul x 10 BSA

37C 2hrs.

I have also rtied digesting less DNA with 0.5ul enzyme for less time. Still the same! I have always set up reactions like this with no problem until now!

thanks for your help





have you changed digestion buffer? Could the buffers be contaminated?

Can you list your digestion mix? Perhaps we can spot something there. Star activity for HindIII, PstI and BamHI is rare if the enzymes and the correct buffers used at the right concentrations are use.
[/quote]

Hello there,

I have tried two different enzyme suppliers (both fresh enzymes each time). The uncut plasmid looks as it should - three forms seen and runs further than the cut plasmid. The plasmids are being purified with the Qiagen mini-prep kit - they are v pure (A260/280 = 1.94 and A 260/230 = 2.36).






I don't think it's star activity because the BamHI band is larger than what you see with the other enzymes, and star activity would produce smaller bands. Also, I think you are loading too much marker, because the bands are far too 'smiley', especially at the top of the gel. If you're having trouble seeing things, stain for longer.

Step 1 would be to repeat the experiment with someone else's enzymes. Next, I would suggest you re-precipitate your plasmid again.

What does the undigested plasmid look like on a gel? For t hat matter, how do you purify the plasmids in the first place?
[/quote]

Repeat the experiment with a fresh tube of enzyme and buffer if you suspect these are not good.
Load more DNA than you are currently use to a gel to get a better picture.
Star activity shows up when you use too much enzyme, you did not seem to use that high amount.
Not all enzymes have star activity. BamH1 may not have such activity. Check with NEB for RE properties.

You are diluting the DNA prep minimally with this reaction mix. In general, lower concentrations of DNA work better for restriction digests. This also dilutes contaminants, see below. I'd recommend doing this digestion in 100 ul as follows:

5 ul DNA (700 ng at your concentration)
10 ul 10x buffer
10 ul 10x BSA (are you sure it is 10x? BSA is often shipped at 100x, then you should use 1 ul)
1 ul enzyme
74 ul water

Digest 1 hour -- digestion is faster than most people realize. You may want to heat kill the enzymes at 65 or 80 degrees for 20 minutes, depending on your next step.


The reason dilution is important is to reduce the effect of any contaminants in your DNA prep. This could be alcohol, phenol, EDTA, whatever. But you don't want them. If I had trouble with this prep, I'd ethanol precipitate my DNA and retry the digest. Another possible problem is the water you are using. Make sure it is of high quality (milliQ etc.)

You haven't said what this digest is for, but if it is for cloning, then these concentrations that result are fine. For other applications, you may have to concentrate the DNA following this step.

Hello,

Thanks for the advice. I will try to re-digest diluting the DNA prep more - although I have tried digesting less DNA (approx 2-3ul of plasmid prep in 20ul final volume).

The BSA I use is 10 X - I dilute the 100X supplied by 10 for my stocks. The resulting digested DNA is for cloning.

What puzzles me the most is that I've been doing these digests for many years - seemingly under the same condtions without any problems.................

Thanks again





You are diluting the DNA prep minimally with this reaction mix. In general, lower concentrations of DNA work better for restriction digests. This also dilutes contaminants, see below. I'd recommend doing this digestion in 100 ul as follows:

5 ul DNA (700 ng at your concentration)
10 ul 10x buffer
10 ul 10x BSA (are you sure it is 10x? BSA is often shipped at 100x, then you should use 1 ul)
1 ul enzyme
74 ul water

Digest 1 hour -- digestion is faster than most people realize. You may want to heat kill the enzymes at 65 or 80 degrees for 20 minutes, depending on your next step.


The reason dilution is important is to reduce the effect of any contaminants in your DNA prep. This could be alcohol, phenol, EDTA, whatever. But you don't want them. If I had trouble with this prep, I'd ethanol precipitate my DNA and retry the digest. Another possible problem is the water you are using. Make sure it is of high quality (milliQ etc.)

You haven't said what this digest is for, but if it is for cloning, then these concentrations that result are fine. For other applications, you may have to concentrate the DNA following this step.
[/quote]

Hello again,

Another picture

This time I digested 3ul in 30 - not quite 5 in 100 but should still be OK no? My new theory is that there is a shift goind on some-how - maybe protein sticking to the HindIII ends? whcih I why the smear looks like a higher mol weight? I'm trying w/o BSA.

Any thoughts on my theory?


Sulked out





You are diluting the DNA prep minimally with this reaction mix. In general, lower concentrations of DNA work better for restriction digests. This also dilutes contaminants, see below. I'd recommend doing this digestion in 100 ul as follows:

5 ul DNA (700 ng at your concentration)
10 ul 10x buffer
10 ul 10x BSA (are you sure it is 10x? BSA is often shipped at 100x, then you should use 1 ul)
1 ul enzyme
74 ul water

Digest 1 hour -- digestion is faster than most people realize. You may want to heat kill the enzymes at 65 or 80 degrees for 20 minutes, depending on your next step.


The reason dilution is important is to reduce the effect of any contaminants in your DNA prep. This could be alcohol, phenol, EDTA, whatever. But you don't want them. If I had trouble with this prep, I'd ethanol precipitate my DNA and retry the digest. Another possible problem is the water you are using. Make sure it is of high quality (milliQ etc.)

You haven't said what this digest is for, but if it is for cloning, then these concentrations that result are fine. For other applications, you may have to concentrate the DNA following this step.
[/quote]