picking up colonies and growing in the broth after transformaion - cloning with competent cells (Jul/19/2011 )
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
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.