I have been trying to make a very simple construct for many months
and can't find out where the problem is! If it doesn't work soon I don't know what I'll do...
Here is what I've tried:
Vector DNA: 7 kb : Single digest with Age I, gel purify.
Insert: 1.4 kb PCR product, PCRd with primers that have AgeI linkers added (5 bp overhang on each end)
Protocol: Digested vector and insert with Age I. CIP’d vector and gel purified. Gel purified insert.
Ligated at 2-3:1 molar ratio of I:V in 10 ul with 40 ng vector. Transformed 5 ul into 100 ul NEBalpha cells.
Results: Same number or less colonies on V+I as V.
This has been repeated many times, with the following variations:
Age I Enzyme tried:
Promega Age I – still had some uncut but ran gel a long way.
Biolabs Age I – still had a small amount uncut.
FastDigest Age I – did not see any uncut after digestion. (Used at least 5x excess of enzyme and digested up to 4 hours)
CIP tried: Promega, Roche (gel purified after; did not heat inactivate)
Ligase tried: Promega, Biolabs. Ligations carried out 16 C overnight.
For the insert, I also put the PCR product into the Zero Blunt vector and cut it out with Age I and gel purified the 1.4 kb band – so I know the insert was successfully cut.
DNA fragment purification methods tried:
Qiagen gel and PCR kits (eluted with H2O);
Roche glassmilk (gels) and PCR cleanup columns (eluted with TE or Tris pH 8)
Also bought in some BioRad Mol Biol grade agarose for gels and HPLC water to use for enzymatic reactions.
I also found a different prep of the vector I had made in a dam- host and tried that.
V only - quite a few colonies (a couple hundred colonies on a 10 ul of 100 spread onto plate)
I only - no colonies
V+I – same number of colonies as V only
I always get Vector only background and nothing else. The Ampicillin is working because when I transformed a control construct I got a much higher no of colonies.
I think there is a problem ligating the V+I but have no idea how to troubleshoot this any further.
Is there any chance my competent cells don’t like the 1.4 kb fluorescent tag I’m putting in? The tag is Green Cherry. The construct I’m trying to drop it into has been used successfully before to make other tagged versions like mCherry.