I am trying to do gibson assembly cloning. I have two fragments for gibson assembly cloning (plasmid backbone and insert). After gibson assembly reaction I have transformed this in E. coli. DH5alpha and grown this transformed bacteria in luria agar plate containing kanamycin as the plasmid I am using contains kanamycin resistance gene. After 2 days I got very small colonies. From this colonies I have done colony pcr from 5 colonies for my insert and I got bands from these colonies. From this positive colonies I have again grown these bacteria in fresh agar plate containing kanamycin. In the same way I got the colonies after 2 days and have done colony pcr again and have not found any band for insert and also there is no growth of bacteria after adding these bacteria in liquid Luria Broth. I want to know what are the possible reasons behind this.
Can anybody help me with their suggestions?
How much kanamycin you were using? I usually used 37 ug/ml and have a 'good looking' colonies after overnight incubation
You need to pick colonies and streak them onto a NEW plate before you can do colony PCR. You are probably getting false positives in your PCR due to plasmid that is on the surface of the plate (and not transformed into the cells) on the original selective plate.
Good luck!
Two days is a long time (assuming growth at 37). What happens if you transform in a control plasmid (like pUC19)? Does it still take 2 days? If so, it might be time for a fresh batch of media.
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