Yesterday's experiment worked very well, in that the thorough controls clearly tell me where the problem is. But the actual experiment produced only three candidate colonies.
Control E: No DNA. No SpecR or AmpR colonies. GOOD-selective plates kill non-resistant cells
Control F: AmpR SpecR Plasmid. p∆TA::Spec: ~350 AmpR and ~350 SpecR transformants GOOD-selective plates select for cells carrying the resistance genes on a plasmid, and the competent cells transformed efficiently.
Controls D and G: EcoRI-cut pUC18 ± ligase. ~12,500 AmpR colonies after ligation, only about 250 without ligase. GOOD- ligation worked.
Control C: No-ligation control. Kinased spec PCR product plus not-kinased inverse-PCR product, ligation reaction with no ligase. No SpecR or AmpR colonies. GOOD-The fragments do not spontaneously circularize and transform cells, and the fragment mixtures do not contain any unwanted intact plasmid.
Control B: Kinase control. Ligation of kinased inverse-PCR fragment. Should have given AmpR colonies, but none. BAD- Kinase failure.
Experiment: Ligation of kinased spec PCR product plus not-kinased inverse-PCR product. Gave only 1 AmpR colony and two SpecR colonies.
I've streaked the three candidate colonies on both Spec and Amp plates. The desired plasmid should confer resistance to both.
And I looked at the expiration date on the tube of BioLabs T4 polynucleotide kinase I've been using. 03/09!!!! AAARRRGGGHH!!!!
Have I been using the wrong tube of kinase? Is this not the kinase that the former undergrad and sabbatical visitor used successfully last year? Searching the 'Special Enzymes' freezer box turned up another tube of T4 polynucleotide kinase, but this one looks even older. So I've just emailed the undergrad and sabbatical visitor to ask what they used.