The test experiment was to kinase, ligate and transform into DH5alpha the product of the inverse-PCR reaction. If the T4 polynucleotide kinase reaction worked, its blunt ends would acquire 5' phosphates that would allow it to be circularized by T4 DNA ligase, and to then transform DH5alpha to ampicillin resistance. The negative control was no DNA and the positive control was the same p∆TA:spec plasmid that had given thousands of AmpR and SpecR transformants in the previous experiment.
Sounds great, but this time the positive control didn't give any transformants at all! Background small colonies were frequent, possibly because the plates were a bit old and the ampicillin had lost its potency, so I didn't trust the few larger colonies on the inverse-PCR reactions plates. I streaked a few of the large colonies to check if they were genuinely AmpR - one was.
I repeated the control transformation and negative control with new Amp plates; the no-DNA control plates were clean but so were the p∆TA:spec plates.
I thought the problem might be the plasmid, but I wasn't sure I have another reliable positive control. So I did a miniprep from the one genuine AmpR colony I had streaked and transformed the cells with that DNA. I also had the usual no-DNA control, the undergrad's p∆TA:spec, another plasmid made by the undergrad (used successfully by me as the inverse-PCR template, and some pUC18 left by a sabbatical visitor.
Success all around. The miniprep DNA, the other undergrad plasmid and the pUC18 all gave lots of transformants (the photo shows part of a pUC18 plate), and the undergrad's p∆TA:spec and the no-DNA control gave none. I don't know why the p∆TA:spec plasmid worked well in my first experiment - maybe I had grabbed a 'wrong' (i.e. good DNA)tube.
Next step, repeating my original experiment (the one in the previous post), this time with better controls.
- DNA clean-up: I did a new inverse-PCR reaction because the old one got used up in the tests. I need to start by doing a spin-column cleanup of it.
- Two kinase reactions: (i) the 'blurry' spec PCR product and (ii) the not-blurry inverse-PCR product. Heat-inactivate the kinase before step 3 (65°C 20 min). This time I'll use a newer stock of ATP, and the official kinase buffer.
- Four ligase reactions: A. The kinased spec fragment plus not-kinased inverse-PCR fragment. B. (kinase control) The kinased inverse-PCR fragment. C. (negative control) The not-kinased spec fragment plus the not-kinased inverse-PCR fragment . D. (positive control) pUC18 cut with EcoRI and heat-inactivated (65°C 20 min).
- Six transformations: Ligations A, B, C and D, plus 1 µl pUC18 as positive control and no DNA as negative control.
Preparations: We have enough kinase, and I've just sent the grad student to buy more ligase. Luckily I have lots of frozen competent cells for the transformations. I'll need to digest the pUC18 and check it in a gel, and pour lots of Amp plates and some Spec plates.