Dear All,
I am facing problem with commonly used expression strain E.coli BL21DE3. With transformed plate, I picked up single colony and inoculated in 10mL LB media (with Kanamycin 30ug/ml). Next day from overnight grown culture, I seeded fresh 10 ml LB media (with Kanamycin 30ug/ml) at 0.2 OD/ml. But it took around 4-5 hrs to reach 1 od, which is highly unexpected.
When I checked plasmids in those cultures they were perfectly fine.
Sometimes I also found some thread like structures during this incubation.
Kindly suggest any solution if you have...
Dear Shahanoor,
Thank you for your kind ans. Actually in our lab we use to inoculate 10 ml media in 50ml of tube, which is already fixed and normal protocol for the lab. And everytime it's normally growing.
I will definitely try warming the LB before use..
Thanks.
Hi Asha you can try the enriched culture mediums (ex. super broth or terrific broth) instead of just LB media, for helping bacterial growth. Sometime plasmid contains too many proteins that slowdown the growth rate of the bacterial cells due to they work too hard.
Wish you good luck.
Hi All, thank you very much for your valuable suggestions.
I would like to update you all regarding my solution. I got my answer.
Actually the Plasmid which we were using was bit toxic to the cells. therefore whenever, I used to inoculate the LB+Kan media with colony/culture, and incubate it overnight, culture will be in late stationary phase next day. So If I am considering next day morning OD as 100% viable cell OD and seed next flask at 0.2, It won't be actual 0.2 OD. There will be hardly 0.001/0.0005 OD (viable cells). So it used to take longer time to reach 1 OD.
So to solve this, I tried to grow cells at lower temperature (25c), so next day its stage won't be later stationary any more. And I will be having more no.of viable cells in my micro-pipette tip.
Thank you again.
Regards,
-Hitarth
Intas Biopharma
Ahmedabad, India.
Ph. 9428316389
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