I was trying to cut a piece out of an existing plasmid and replace it with a promoter of my own. However, the RE sites on the plasmid are limited. The only thing that will cut at the 5' end is AfeI, which happens to be blunt. For the 3' end I picked BamHI.
Then I digested the plasmid with those 2 enzymes overnight, PCR'ed and digested the promoters with the corresponding REs. I also purified both digestion products using gel extraction. The vector end up being around 7kb while the insert is around 2kb. However after ligation and transformation, no colony appeared. I'm pretty sure the transformation is working since positive controls all look normal.
I tried both the Fermentas CloneJet and T4 DNA ligase for the ligation. I used 25ng of vector and 75ng of insert and ligated at 14 degrees for up to 24 hours or at room temperature for 1 hour. Neither strategy worked. Then I doubled the ligase concentration but still saw no colonies.
Right now I'm back to square one: is it even possible to ligate blunt and sticky ends at the same time? Should I just blunt down the BamHI site with Klenow and do a blunt ligation instead? Or would it be more time-efficient resorting to some other method (such as PCR fusion)?
Any hint would be helpful!! Thanks in advance~!
Edited by sanguinara, 30 July 2010 - 02:33 PM.