I recently got plasmid from Addgene, however, I failed to expand them into glycerol stocks within 2 weeks, as recommended. It had been two months since I got the plasmid, and in the middle our 4 degrees freezer had also broken down. So I suspect is not usable anymore. I nevertheless plated the bacteria as I could see something growing in the agar stab. But after 24 hours, I do not see any growth. May I just confirm (from those who have used addgene plasmids) that 2 months post receipt of the plasmids, they will no longer be viable?
Thanks and Regards,
Mathangi
My guess is that after sitting at 4 degrees (plus warmer conditions) may have caused your stab culture to die. You try streaking them out some more in the hopes that a few cells are viable. I've had bacterial plates of E. coli still show viability beyond 2 months but they were cold the entire time.
Amy Klocko Ok thank you for the suggestion! So you mean I should try streaking from the stab culture again right? I will nevertheless place an order for a fresh stock
You can try and restreak again to see if you get growth, but it may well be the strain is dead. The lifespan of E. coli stored as you did will vary dramatically depending upon whether the host strain is RecA+ or RecA- (and most cloning strains are RecA-)
However all is not lost. If you can recover some of the cells from the stab or the solution, even if dead, you can do a mini prep on those cells and retransform the plasmid into fresh cells. Just because the cells are dead doesn't mean the plasmid DNA is not intact. I've done this a number of times from a colony that will no longer grow or a dead stab.
I agree with trying a re-streak from the stab, and with trying a plasmid prep and re-transformation with the cells (even if they are dead). It can work!
Amy Klocko and Michael J. Benedik thank you so much for the advice and I will follow the steps!:)
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