recombinant plasmids lossing - get HELP! (Nov/03/2007 )

Can anyone do me a favor to slove this problem that:

I had constructed a recombinant plasmid (pET28a-Hly) and transform BL21(DE3). The first time I try to induce the recombinant strain, I've got lots of particular protein, of course by SDS-PAGE; then I store it (strain) -20c with glycerol(15% concentration). But, some days later, now, I can't get that much protein than before, somebody'd telled me that maybe recombinant plasmids will loss in the period of strain storage, or inoculated so many times will cause plasmid-lossing also. I've got puzzled.

Thx a lot for anyone that could solve this problem in my head

a fresh bio-researcher nuclease

BL21 has full functional endonuclease and recombinase pathways (endA1, gyrA96(nalR), recA1 and relA1 are all functional). As a result whatever plasmids kept in said strain will be damaged/rearrange given time. Furthermore, if you kept a sample of your culture after inductions, there is a strong probability that a proportion of the cells in said culture would have lost the plasmid.

I would recommend that you retransform the BL21 line with the desired plasmid and go from there.

thanks for your help perneseblue, i got it, and here're some more questions--

you mean that recombinant plasmids will be digested by endonuclease in BL21, and will the endonuclease or something like that exist in DH5a? I had constructed a plasmid containing that fragment with pMD19-T (a clone vector). Will this plasmid loss in DH5a?

if i want to store my plasmid (pET28-hly, i'd described before), should i store it in DH5a?

In DH5a, the endonuclease endA1 and recombinase recA1 have been knocked out. DH5 is a cloning strain rather than an expression strain.

As for plasmid storage, I believe in storing plasmid DNA rather than in cells. DNA is more stable, won't die, large quantities can be stored. and can be kept at -20 Celsius. People do keep cells (containing plasmid), I am just not one of them.

I Agree with Pernesblue a 100%. If you have plasmid DNA you can always multiply in DH5a or JM109 with just 2ul of plasmid.

That's very kind you, perneseblue, thanks!

i'll do as what you say. Store plasmids and use Dh5a as a competent cell to maintain my plasmids.

plasmids seems to be more stable than recombinant strain, isn't it? what medium should i chose? TE buffer or just dd water? water is surely convenient, but somebody'd told me that TE was better because plasmids will degradate in water in a long-term storage. is that true?

btw, does 'recA+ ' means 'has recA'? is 'recA' same to 'recA1'? and the same question about 'endA', please tell me.

nuclease