A mystery!!! Plasmids smear when digested with site-specific enzymes

[doug1c]

This is my first time using this forum and also my first post. I'm hoping that someone out there can help me out and explain why I'm seeing what I'm seeing.

For my cloning, I have been using PMC103, a e.coli strain that is deficient in recombination. For the past couple of years, I have had no problems with this strain and was able to clone and also reclone vectors from this strain. However, lately, I have been noticing that whenever I transfect plasmids (not just ligation reactions) into this strain, grow them up and miniprep the plasmid derived from this new strain, the plasmid smears whenever I tried to digest them with site-specific enzymes. (I didn't over-digest and checked for star activity) I have done this with several types of plasmids, different combinations of enzymes and have also sequenced the plasmid to make sure that the plasmid did not recombinate. Even though the sequence data is good, plasmids derived from this strain always produces a smear. I've also made newly competent cells for this strain, and the same smearing phenomenon is produced.

The really interesting thing is that when I take plasmids (the ones that always smears) from the PMC103 strain and then transfect them into a different strain, such as STBL4, the plasmid is normally digested and does not smear. Can someone explain this to me?

[jlf]

Did you miniprep with too many cells? If you use too many cells, the lysate will be very viscous and a lot of the chromosomal DNA will shear and contaminate you plasmid. When you digest with restriction enzymes, chromosomal DNA will cleave at different lengths and manifest as a smear.

[mutantmutant]

I had this same problem with Stbl3. Stbl3 and PMC103 are not endonuclease deficient (endA-). You end up with a bit of endonuclease activity in your plasmid, then when you incubate at 37 degrees for your restriction digest, your plasmid gets chopped up in random places as well. The solution is simple: you must do the "optional" wash step in your miniprep kit that comes before the {70% ethanol + salts} wash. For example, in the Qiagen miniprep kit, you must do the 0.5 ml PB wash before the 0.75 ml PE wash. If that still doesn't work, try heating the PB to 50 degrees or doing two PB washes. The chaotropic salts in PB (or equivalent) will denature the residual endonucleases.