Hello everyone,
I am having a trouble in keeping my plasmid inside the bacteria. My plasmid is a Lentiviral plasmid of 7 kb and with the insert that I cloned is now 9 kb. The resistance gene is against Ampicillin. After cloning I transformed the ligation product into Stbl3 bacteria and had low yield of plasmid (15 ng/uL), then I tried DH10B and after first passage it was nice (240ng/uL) but then when I do glycerol stock for -80 degree, the yield was very low again (5ng/uL). Then I tried NEB Stable and the first passage was nice again (430ng/uL) but after -80 stock, I am getting again low yield (10ng/uL)...
So I don't know what to do, to have high yield of plasmid every passage..
If anybody has faced with this problem can share the knowledge it would be very nice.
Thanks!!
Have you tried growing your culture on solid media before doing it in liquid?, I frequently run into issues when I inoculate liquid media directly from the glycerol stock.
Have you tried to streak out from the -80 stock to get single colonies on amp plates and then pick a colony to grow for your plasmid?
Yeah, I am trying it. Tomorrow I will pick some colonies but I dont want to spare 1 more day before every passage. Thats why I wanted to ask If there is any opinion or advise.
Thank you so much.
The problem often is that when you grow a culture in ampicillin, the antibiotic is quickly degraded so for the final few hours of growth of the culture there is no selection. You probably used a dense culture to prepare your glycerol stock, and if there was a strong selection against the plasmid, then some substantial fraction of the population is plasmid free or carrying some sort of deletion. You might try to pick a single colony and grow up to mid log phase (and not overnight) and make glycerol stocks from that. Those might work better for you.
Then it should be the case for all Amp resistant bacteria, right ? Just for curiosity because this is the first time I am facing with...
Thank you!
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