Confirming insert???? - (Dec/01/2011 )
I have done transformed the ligated product into the E coli DH5 cells the colonies were growing on the LB agar with ampicillin plated but when i inoculate them onto LB broth with ampicillin for plasmid isolation they were not growing at all.in most of the tubes I use 15 ml test tubes with 5 ml broth and grow them in shaker.
what could be the problem???
Later on i got Some growth in some broth from them i isolated plasmid and Subjected it to RE digestion iam getting almost double sized insert I cold not understand what is happening
I use BamHI as one of the enzyme and digest for 2 hr.
can anyone suggest me on this regard so that i can confirm my insert .
did you put proper controls to make sure your plates and competent cells were free of contamination?