Plasmid doubled in size??

[CLICchick]

Hi

I'm working with a pET vector that should be 6.1 kb with my insert. Initially, I had some trouble with our cloning strains (XL1 blues) and was running out of plasmid, so ended up transforming BL21 DE3 pLysS cells. I miniprepped from those but am now seeing something strange. When analysed on a 1% agarose gel, I get 6 bands (way more than I'd expect from supercoiling) and, when linearised, shows a size of between 10-12kb.

Is is possible that the plasmid was somehow recombined by the BL21 cells? Why is it double the size it should be?

[mmorgan]

Hi,
I believe that some strains of bacteria used for protein expression (BL21 is one I think) have a high plasmid DNA mutation rate. I know that the strain I use (A BL21 derivative) is not recommended for doing cloning in so I only transform the plasmid right before I do the protein expression experiment.

So it's possible that the bugs have scrambled your plasmid a bit. Does the protein expression still work?

[JennYeap]

BL21(DE3) expression host contains a phage construct that expresses T7 RNA polymerase under the control of a lacUV5 promoter. Most likely you have isolated the phage instead of your plasmid.

[SAFIA]

better use dh5 alpha

[JennYeap]

alternatively, u can use BL21 cloning host, XL1-blue, NovaBlue or Top10 cells.

[hairongxu88]

thanks