I made 30 bacteria transformed with plasmid including ampicillin resistant gene.
transformed bacteria (total 30) were mixed with 25% glycerol and stored in -70 defreezer ----> stock : 500 ul LB with bacteria + 500 ul glycerol (50%)
but, during a experiment, mistakenly , i left one of bacteria-stocked tubes on my table for 2~3 days.
when I picked up other 29 bacteria tubes from defreezer and grew them in LB broth, they grew well.
However, the remaining one bacteria that was left on my table for 2~3 days does not grow anymore. now, I need to grow the bacteria ....
is there any reason why the bacteria does not grow anymore ? I think i made glycerol stock well because I successfully grew other 29 bacteria stock in LB broth.... but only the bacteria tube that was left on my table for 2~3 days does not grow anymore..
lefting for 3days on my table made the bacteria killed ?
thank you for reading my question !
if there is no way to grow the bacteria, i will extract plasmid from the bacteria stock and transform the plasmid with new competent cell...
I'm not certain but I would guess that the osmolarity is too high in 25% glycerol and so they died after leaving on the bench. I would suggest just doing a mini prep on some of the cells from the stock and retransforming.
thank you for the answer! yesterday, before I go to home, I inserted all remaining bacteria suspension into LB broth and grew overnight with shaking. a minute ago, I checked whether bacteria grow or not and I noticed that bacteria finally grow.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning