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picking up colonies and growing in the broth after transformaion


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#1 oms

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Posted 19 July 2011 - 10:07 PM

I finally could manage to use my 3.5 kb PCR product in a bicistronic cloning vector pIRES2-AcGFP1 and transform onto stellar competent cells (clontech) and plated it on LB agar+kanamycin(50ug/ml)+X-gal. After a struggle of 3 months I could see finally white colonies on the plates. From these I picked up 20 colonies and transferred onto LB broth for further plasmid extraction. To my surprise all the 20 tubes were absolutely sterile. I again picked up 20 more colonies the next day and transferred to the broth+Kanamycin . Much to my surprise no growth. I then thought there must be some problem with the media that I have prepared so I borrowed the media from other lab and agin picked up 10 more colonies, again no luck..no growth. I even picked up a colony and streaked onto a fresh plate of LBagar+kanamycin.
No growth. What does it mean? What is it then grown on my plate? can anyone explain me this?

Thank you in advance

#2 Gangwolf

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Posted 20 July 2011 - 02:12 AM

It could be that the kanamycin in agar plates is either in too low concentration or too old.
Try use a new batch and increase the concentration.




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