Difficult Ligation - Details Inside - (Jan/13/2013 )
XbaI is dam sensitive. Spe1 is compatible with Xba1. Maybe, use klenow and ligate blunt end. Did you do Re-cutting? PCR with Taq or Pfu? Generally, try different ratio for ligation.
I already changes my mistake. Sorry.
Maybe blunt...what do you mean re-cutting? And we do our PCR's with half Taq and half Vent. I may try a 3:1 ratio...
Better to increase insert. On your Gel you dont see your expected size? Maybe difficult on this size of an insert. With recutting i mean where do you no that its not working?
I do see the right sizes on the gel. And it's not working because I haven't been able to get any transformations. I guess technically that doesn't mean anything, but I find it hard to believe I would have a working ligation without transformations.
I guess you use Calcium Chlorid and using all tranformed E. coli to plated on Amp plates.
Calcium chloride can increase the efficiency of a transformation, but depending on your e.coli cell type I would try throwing in some BME. When I was cloning a vector + insert around that relative size, the carbohydrates on the e.coli's outer surface were prohibiting proper entry. 2-4uL/100uL of cells should do the trick. have you tried confirming your insert post-ligation? Just try PCRing your vector + insert (find a primer within your insert + a primer outside, just to make sure you didn't ligate a doublet or triplet into your plasmid).
When you run a control with your cut plasmid, do you get any colonies? With your original plasmid? Check the efficiency of your e.coli cells with a stable plasmid.
Hope this helps