Miniprep from BL21(DE3) - (Jul/26/2007 )

Dear All,
I seem to have lost the plasmid DNA of one of my clones which I would like to perform some PCR deletions on. I do have a glycerol stock of BL21(DE3) cells transformed with the clone though. Would it possible to a inoculate a culture with the glycerol stock and miniprep fresh plasmid DNA from the BL21(DE3) culture. As a consequence of the BL21(DE3) host should I expect any modifications on the DNA. I imagine yield will not be as good as that from an amplification strain such as dh5alpha but is this the only drawback. I really need this clone and I have tried to redo the mutagenesis to make it again but I can't get it to work.

Cheers

Yeah, I suppose you can miniprep the BL21 cells. The only thing is that these cells are not the most appropriate to store plasmids, but I suppose that at -80ºC the chance of the plasmid having mutations is not so high. If they were at 4ºC I wouldn't trust it very much...

I say give it a try if it the only option you have.

I recommend that you inoculate some other strain with that plasmid for storage though.

Thanks for the reply and I'll give the miniprep a try.
You made me wonder why BL21 cells were not good for storing plasmids?.
Cheers,
Sat

As already mentioned by Ambrósio BL21 is not the best thing to keep your plasmids in. I would strong suggest:

1- a tiny bit of glycerol stock be restreaked on a plate. Once single colonies have grown up, conduct a miniprep on several colonies ( try 10) Assume high instability, and grow your cells at low temperature ~28 to 30 celsius, and don't over grow far past saturation.

2- Extract fresh plasmid DNA from the culture and characterise the plasmid. Once you have the right plasmid, move that plasmid into a cloning strain. Either make frozen stock of these cells or extract more plasmid DNA and store the DNA instead.

As above. Two difficulties occur. BL21 strains are recA+ and recombination is common. Second, they are endA+, which means that DNA you purify during a miniprep is often damaged by endonucleases. Transform the DNA promptly into a cloning strain, and don't try to store the DNA sample for long periods. You should also do the suggested second wash with PB for endA+ strains (see your miniprep manual).

Thanks for clarifying.
Sat