I'm pretty sure that I've now done all of the cell preps for our big planned RNAseq analysis, more or less as diagrammed in the previous post.
Instead of a cya knockout mutant as a negative control (pink in the diagram I used a crp knockout. cya encodes the enzyme that synthesizes cyclic AMP (cAMP), and crp encodes the transcriptional activator CRP, whose ability to induce transcription is entirely dependent on cAMP, so the two mutants have the same phenotype - inability to induce both the competence genes and the energy-balance genes in the CRP regulon. I decided to use the crp mutant partly because that strain grew up first and partly just in case there are traces of cAMP in our sBHI medium. I did one MIV-competence time course with this mutant (4 samples) and one sBHI time course (3 samples rather than the 2 in the diagram).
I also did 3 sBHI-timecourse samples of the sxy knockout as another negative control (also pink in the diagram). I think I now have two more samples than will fit in 3 lanes of sequencing (24 samples multiplexed per lane), so I'll probably omit the OD=0.6 samples of the negative control sBHI timecourses. But I'll process the RNA from them just in case something goes wrong with another sample.
I found my missing DNase. I hadn't lost it, just ordered the wrong kind. So now I have $250 worth of a high-quality DNase I don't really need, and will need to order the right kind.
I'll get to the RNA preps once I get some teaching responsibilities under control - partly the grading for my face-to-face Human Ecology course but mainly the need to rerecord ~150 lecture videos for Useful Genetics/Genetics for Life.
Saffron - pricey condiment
4 hours ago in The Phytophactor