Is it possible to ligate blunt and sticky ends at the same time? - newbie mistake... Please help!! (Jul/30/2010 )
NemomeN007 on Wed Aug 11 03:27:02 2010 said:
Well...it's a long shot, but without a map and sequence, there is a chance that you're removing an important part of the backbone..either part of the ori or part of the resistance marker/promoter region which will result in no colonies after transformation....
Thanks for the input! I do have a (partial) map so I'm partially confident that no important parts are cut out.
perneseblue on Thu Aug 12 04:13:31 2010 said:
If the insert and vector were gell purified, there is no need for phenol-extract step. The DNA is clean. If you do phenol-chloroform extract, ethanol precipitate the DNA, and wash the DNA pellet in 70% ethanol. Phenol can inhibit down stream enzymatic reactions.
If the 1-blunt end/1-stick end ligation strategy does not work, you might want to consider a different strategy. Perhaps consider PCRing the vector to add your desired restriction site. Both vector and insert can be modified by PCR.
I'm on it! I managed to ligate the insert into a Clonejet vector after blunting it. Hmm. I'll work on the vector now.