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Antibiotic not working in LB agar, fine in broth

antibiotice.coli cloning transformation

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#1 TK04

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Posted 27 August 2021 - 04:01 AM

Hi there

 

A few weeks ago I plated my E.coli DH alpha cells after transforming with my golden gate reaction. This had worked fine for a year using the same batch of competent cells and antibiotic. However, on this occasion, the kanamycin LB agar plates all had a lawn of E.coli as if the kanamycin did not work. My control plate with kanamycin (with untransformed E.coli) also had a E.coli growing. I thought my kanamycin stock which had been at -20C for almost a year had gone bad. i made a fresh stock and i had the same problem. I keep the kanamycin powder in the fridge as per manufacturers instructions.  I then thought i had contamination from Kan resistant e.coli from one of the transformations. i autoclaved everything possible, but still. i then used my fresh antibiotic stock in LB broth and inoculated with untransformed E. coli and the kanamycin worked fine, not bacteria grew, and the control without Kan grew. Why is it that the kanamycin is not working in LB agar but fine in broth?



#2 bob1

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Posted 28 August 2021 - 02:54 AM

Heat - check your waterbath temperature, ensure that your agar is cool enough before adding the antibiotic.



#3 TK04

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Posted 30 August 2021 - 05:44 AM

Heat - check your waterbath temperature, ensure that your agar is cool enough before adding the antibiotic.

The heat seems fine. i cool the medium to temps i am comfortable touching the bottle in my hand. i have done it that way many times and it has been fine. it only started giving problems recently.



#4 bob1

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Posted 03 September 2021 - 02:09 AM

How old is your Kan powder? - get a fresh bottle in.







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