how to clone blunt end and sticky end simultaneously? - (Jun/11/2006 )
i am cloning a TAG in pCR3.1 VECTOR ,having one end blunt and one sticky,after transformation in E.coli, colonies appeared on the ampicllin plates but not showed any growth in the same selected broth.
at what temperature did you do the ligation step?
14 c overnight
16 c overnight
4 c overnight
When I have to ligate sticky and blunt ends, I always use the conditions suggested for the blunt ends.
Did you prepare fresh plates where you plated the transformed bacteria?
yes i always used the fresh plates.but whats make difference if we use the older one?
I was just wondering if the ampicillin of the plates was inactive, because it was the only explenation I could think about for the colonies growing on a plate but not in culture
we checked the ampicillin plates and also controls are with the experiments .problem is not there.is there any other possibility?
whats the role of salts in ligation product?inhibit ligation?