Still no cloning success

Here's what I've done and what I've learned:

At the end of my last post I was about to test the toxicity to E. coli of the 'toxin' gene adjacent to the gene I'm trying to knock out.

I did this by instead trying to create the same plasmid the undergrad had created, one that (unintendedly) deletes the end of the toxin orf as well as the adjacent gene.  So I repeated the inverse-PCR reaction using her old primers instead of my new ones, and used this fragment in my kinase-ligate-transform experiment.  I tested both the ability of this fragment to self-ligate after being treated with kinase, and its ability to ligate to a SpecR PCR fragment that had been treated with kinase.  The former produced only four AmpR colonies, and the latter no AmpR SpecR colonies.  I didn't do plasmid preps on the AmpR colonies to see if they contained the expected plasmid, though maybe I should have.

Although failure of this experiment does not disprove the hypothesis that the toxin gene is toxic to E. coli, it does disprove the hypothesis that the hypothesized toxicity is the reason that my previous experiments failed.  Sorry, that's a nasty sentence - try again: Since this experiment worked for the undergrad last spring but not for me now, toxin-toxicity is unlikely to be why my experiments are failing.

Next I made a list of the various approaches I could try - I'll get back to these below.

The next test I did was to see whether I could ligate the SpecR PCR fragment to a plasmid that didn't need phosphorylation, and whether simple blunt-end ligation was working at all.  If this worked I'd know that the problem is the kinase reaction.  So I cut a simple plasmid (pUC18) with EcoRI (Makes sticky ends) and separately with SmaI (makes blunt ends).  Both these cuts leave ends with 5' phosphates that should be good substrates for ligase.  I heat-inactivated the enzymes and set up three ligation reactions:

1.  EcoRI-cut plasmid with ligase
2. SmaI-cut plasmid with ligase
3. SmaI-cut plasmid with SpecR fragment and ligase

Results: 

1. ~3500 AmpR colonies (all of the ligation reaction)
2. 639 AmpR colonies (all of the ligation reaction)
3. 198 AmpR colonies (half of the ligation reaction) and 1 SpecR AmpR colony (other half of the reaction).  But my plasmid miniprep of cells from this colony didn't give any plasmid

As a positive control, the same amount of uncut plasmid gave about 3000 colonies.

So I concluded that my blunt-end ligation reaction conditions were fine.  Can I then conclude that the problem is the kinase reactions?  Unfortunately this wasn't a very stringent test of the ability of the SpecR fragment to be blunt-end-ligated into a vector, because I didn't pay attention to the relative proportions of the vector and insert.  I should have used a limiting amount of the vector and lots of insert, but I actually used about equal amounts.  Since self-ligation of the vector is a unimolecular reaction it is expected to be much more efficient than insertion of the Spec fragment. 

This experiment used up the last of my control SpecR AmpR plasmid stock so I grew up the cells and did a miniprep.  But this didn't give any plasmid at all either, just some chromosomal DNA!  Can something also be wrong with the plasmid prep solutions, or my procedure?

The grad student is trying to use the same kinase to label his DNA with 32P-ATP.  He's not having much success either so he's going to test my fragments, which are much simpler substrates than the sheared chromosomal DNA he's been using.