basic RE digests acting weird - (Jan/28/2006 )

For some reason, I can't seem to do a standard digestion of plasmid DNA (7000 kb). I've isolated different plasmids by Qiagen miniprep (eluting with H2O) and found the concentration by Nanodrop to be in the range of 150-400 ng/ul. Then using fresh NEB enzymes (keeps happening with SfiI, once with NotI, once with BamHI) I do the digestion and run it out on an agarose gel and I see no bands. Sometimes there's a light smearing along most of the lane, sometimes there's just a diffuse band in the 50-100 bp range. This happens when I follow the exact protocol of a coworker who gets normal results. If I run uncut plasmid I see a strong band. I've been mixing the enzyme into solution by pipetting it up and down; is that wrong? What am I doing to screw this up?

Please help me. This is driving me crazy.

HI, it is obvious that you are having degradation of your plasmid DNA.
When this happens there are many variables that you have to consider:
1. did you change the enzyme tube? maybe that specific stock is degrading.
2. did you change the water? do you use fresh autoclaved water? try to use water you only use for these digestions or only for DNA work.
3. are your tips sterile?
4. did you use the same enzymes as your coworker?
5. did you use the same DNA:enzyme proportions as your coworker? sometimes the enzyme starts to degrade DNA when it does not have any more material to cut.
6. have you check if any of your enzymes posses start activity? if so, not over digest. I mean, digest for a time sufficient enough to cut everything but not too much longer. (this goes conected with 5.)
7. in relation to 5. and 6. you can do a test of digestions: prepare several tubes with the same amount of DNA, same amount of enzyme,same conditions and digest for different times, like tube 1=10 min, tube2=20 min and so on. Then run a gel, and check if there is a time that siuts better for you.
8. I was told once, that sometimes you get contamination in your pipettes and you can be contaminating your reactions without knowing. Check this.

Well, I think I gave you enough hints for you to think about.
You can also ask a favour to your coworker and ask him/her to do one tube digestion for you, and check. Maybe it is something in your pipettes or anywhere.

Good luck!!!!!!!
Marcfe



For some reason, I can't seem to do a standard digestion of plasmid DNA (7000 kb). I've isolated different plasmids by Qiagen miniprep (eluting with H2O) and found the concentration by Nanodrop to be in the range of 150-400 ng/ul. Then using fresh NEB enzymes (keeps happening with SfiI, once with NotI, once with BamHI) I do the digestion and run it out on an agarose gel and I see no bands. Sometimes there's a light smearing along most of the lane, sometimes there's just a diffuse band in the 50-100 bp range. This happens when I follow the exact protocol of a coworker who gets normal results. If I run uncut plasmid I see a strong band. I've been mixing the enzyme into solution by pipetting it up and down; is that wrong? What am I doing to screw this up?

Please help me. This is driving me crazy.
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I agree. The DNA is being degraded. You might try a control reaction with RE buffer, DNA and no enzyme. You might have contamination of your DNA sample with nucleases. Try digesting a known good DNA sample with your RE using the same reagents. If that works and your sample does not, I'd suggest phenol/chloroform extraction and ethanol precipitation of your DNA, or, if it is easy, preping the DNA a second time.

You guys were right. I was getting degradation from contaminating endonucleases even though my E. coli strain is endA-. I added the optional wash with PB buffer (guanidinium chloride) in my miniprep and the problem went away. Thanks for the help.