I transformed a library into stbl3 competent cells following a standard protocol which is like incubation 30min in ice, 42°C for 45s, soc culture at 37°C for 1h, before culturing at 30°C on LB Amp+ dishes.
some supplementary info:
1. The dishes look normal after 30°C culture.
2. The plasmid was tested and had no problem.
3. I used another plasmid to tansform and harvested bacteria directly from the dishes(didn't frozen in refrigerator) with correct plasmid.
I can only find one reason for this phenomenon is that -80 made it happened. Can you help me find other factors?
After you are growing the culture for glycerol stock preparation are you directly storing the culture + glycerol ? If so I'd suggest first pelleting down the culture and add fresh media+amp+25%glycerol mix to resuspend the pellet, flash freeze in liquid nitrogen or ethanol dry ice bath and then store at -80 celsius.
Sometimes unstable plasmids are cured after 12 hrs in ampicillin as the half life of amp at 37 celsius is roughly 12-16 hrs. Try using this method or you can try adding carbenecillin instead of amp as its much more stable that amp.
I find it really hard to believe that the plasmid totally disappears from a stock frozen at -80. Are all of the cells without plasmid? Or are there just some cells in the stock that are without plasmid but most are with plasmid?
In order to lose a plasmid you have to have cell division, which is not occurring at -80. So either the stock that was frozen was not actually correct, or you lost the plasmid during the outgrowth from the frozen stock.
Michael J. Benedik You are right. There was a 1.5kb plasmid in the original library (from others), which is much easier to be transformed into competent cells. Therefore, the plasmid I need(~8kb) looks like disappeared.
Aekagra Singh Thanks for advice, I got new skills! Though I have found the reason.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue