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.

-saaam-

at what temperature did you do the ligation step?

-dodosko-

different temp.

14 c overnight

16 c overnight

4 c overnight

-saaam-

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?

-Raffaela-

yes i always used the fresh plates.but whats make difference if we use the older one?

-saaam-

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

-Raffaela-

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?

-saaam-