The meticulous RA thought it would be wise to use PCR to check the genotypes of the purR::kan knockout mutants I used for my time course last weekend. (I had already checked that they were both resistant to kanamycin.) So she designed and ordered some primers that would flank the insertion that was described in the notebook of the grad student who originally made the mutant, and did colony PCR on all four of the strains I had used.
Much to my surprise, all four strains produced bands of the size expected for purR+ cells (about 1.0 kb), and none of them produced bands of the size expected for the purR knockout (about 2.2 kb). Either there's something wrong with the PCR analysis (and she's very meticulous so I doubt that), or the strains aren't what we've been thinking they are.
I had made these strains by transforming cells with DNA I had isolated from cells grown from the old frozen stock of purR cells made by the grad student (at least, that's what I thought I was doing), and selecting for kanamycin-resistant transformants. Could I have used the wrong DNA? Or grown up the wrong cells from the freezer?
We know that the original cells made by the grad student had the correct mutation, both because he had carefully checked them out and because a technician had later thawed a vial and done a microarray analysis of RNA. This showed that the mutant dramatically overexpressed all the genes that were predicted to be repressed by PurR in wildtype cells.
So tonight I've streaked out more cells from the last freezer vial of the original purR knockout, and on Monday the RA will test them by PCR. I also located the DNA I had used for that transformation, so she can test that by PCR too. If these cells give the expected 2.2 kb band, we'll assume something went wrong with my transformation. If they give the 1.0 kb band, we'll carefully check out the new PCR primers and probably run a quantitative PCR of a PurR-repressed gene on RNA from the original mutant and from one of the new mutants (with wildtype cells as control). Or, because the RA has recombineering working well now, she might just remake the purR mutant with her new primers.
If the mutants I used for my time course turn out to not be purR-, I think we'd still be really interested to find out where their kanR cassette is, because we don't have any other mutants with this interesting phenotype. That can be done by cloning out the kanR cassette and flanking sequences (the old-fashioned way or by inverse PCR) and then sequencing the DNA on one or both sides of the cassette.
Friday Fabulous Flower - Squashes & pumpkins
7 hours ago in The Phytophactor