I transformed 4.5kb plasmid with Ampicillin marker into DH5α by electroporation, I need to get the actual number of transformed bacteria on the plate (i.e. one plasmid per bacteria), after transformation I add one ml of media (LB) and incubate for 2hr, then I did dilution and plated on selective media (Amp), I calculated the colony number and I found too many colonies which may not reflect the actual number of the successful transformant but may be because of over growing of the transformed bacteria as I incubated for 2hr during the recovery stage, so knowing the ideal time for bacteria recovery and expressing antibiotic resistance gene would be helpful, any idea about recovery time for the following antibiotics: Ampicillin, Chloramphenicol, Kanamycin and Tetracycline???
Thanks
Hi,
I think 2 hours of recovery is not needed for any of the antibiotics mentioned. Usually, the recommended recovery time for E. coli is one hour.
Actually, increasing the recovery time for an Ampicillin resistance plasmid, will lead to appearance of satellite colonies on your plate.
I would recommend that you use Carbenicillin instead of Ampicillin, since it is more stable and will reduce the occurrence of satellite colonies!
In my hands, I don`t even need to recover cells when I a transforming Ampicillin resistant plasmids. You can transform, ressuspend in SOC medium and plate directly, without recovering (this is for ampicillin/carbenicillin only!!!).
To understand why you do not need to recover ampicillin vector transformations, read the following article: https://bitesizebio.com/10246/fast-track-your-ampicillin-plasmid-transformations/
All the best,
you could use a Petroff-Hausser Counting Chamber, or thoma counting chamber to count each bacterium.
Plating different dilutions of transformation onto drug plate is what i would normally do. Then count for a CFU and transformation eff. cal.
Best wishes,
Charles
Thanks Charles
but my question is about the effect of recovery period!
I personally use recovery times of 20-30 min for Ampicillin, 45min for Chloramphenicol, Kanamycin and Tetracycline (E. coli K12, LB, 37C).
Although these do not represent the minimum recovery time, I can say that for sure that this is a sufficient time. You can refer to those as general guidelines, and if for you it is important to assess the real minimum time required for the resistance cassette expression, I suggest you to make a simple time course experiment:
Transform your cells as usual, then plate 100ul of cells on selective plates at 10min internvals.
If you need to control for bacterial growth/duplication during the same time, you can sample from bacteria that already harbor the plasmid grown in the same conditions (after appropriate dilution).
All the best!
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