Hi researchers,
I am culturing the E.coli after I put the recombinant plasmid. before seeding the E.coli to LB broth plate containing kanamycin, I cultured it for 1 hour at 37C with shaking, then I seeded them in the plate and culture the plate at 37C overnight. however, some plates had many colonies but some are not any. so what are the possible reasons that effect on it ?
if any tips, it will be very helpful, thank you guys.
This is very common problem in cloning. The most probable reason might be the failure of transformation.
Since, you had other plates with colonies, you can simply troubleshoot this problem.
No one can give the actual reason, since it was done by you and you know the process best.
Or no bacteria o sensitive to Km. I will try to see if there are more answerd to it.
If you are using a glass L rod for streaking the plates, then then you must take precautions to streak the culture on the plate, only after the L rod has cooled enough after sterlizing in flame. Using a heated L rod to streak the colonies in a circular manner may kill the cells. this is a common cause of absence of colonies in some plates. However Iam not sure of whether you used a plastic or a glass L rod.
Kamesh Krishnamoorthy Kalyankumar Thank you very much for your advice.
Yes, I am using a glass L rod. Usually, I wait for 20~30 sec to cool the L rod, and maybe I should wait longer. it could be a possible reason. Thank you for your answer.
Abhijeet Singh thank you, buddy, maybe there were some technical mistakes in some plates, I will try again.
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