We are working with E.coli SHuffle. We always kept them at -80 freezer from the purchasing to now. First time, we took a tube one week ago. After transformation, we have no colonies on LB Agar plates (Kanamycin 30microgram/ml). Yesterday, we tried transformation again. And results not changed. We inoculated non-transformed another tube to LB plates without antibiotics. But this plates are empty too. Is there anyone who can help?
Hi Aytan,
Some of my colleagues are working with shuffle T7 strains in our lab, and these - for some reason - sometimes have a very long doubling time. compared to the ~30 minutes of a normal post-lag phase planctonic culture, we can get, in LB, doubling times up to a pair of hours.
How long did you incubate your plates? from ~6pm to ~9am? did you try leaving them at 37c for 4-6 hours more?
for the lag phase in presence of antibiotics, we also had erratic times while using the same vector (but different constructs) so here again, incubation time seems to be the key.
good luck
Hi, Ulric le Paige thank you so much for your answer.
In our previous trials we had 16 hours of incubation. Then we tried incubated cells for 22 hours and it worked.
Additionally, although the expiration date has not passed, cells have lost their competence. After incubation, we made them chemically competent again.
Hi Begüm,
For the competency loss (provided the cells are stored at -80), that is something that happens is you thaw/refreeze an aliquote too many times. I would thus recommend aliquoting your stock of competent cells by "doses" from 1 to up to 3-4 transformations, and discarding what is left after that amount of uses.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating