Here's an attempt to get better focused and organized: (Trigger alert: most of this post is not about the real science but about the tiresome details that get in the way.)
What I've been doing:
1. Preparing for a big mutagenesis project:
I want to isolate a lot more hypercompetence mutants. I have a plan, and I've pre-tested the mutagenesis step, but a key component is a way to efficiently transform rare competent cells in a large population of non-competent cells. The best way to do this is with a cloned or PCR'd DNA fragment containing a selectable point mutation giving antibiotic resistance. In the past I've used a cloned novobiocin-resistance fragment. I had thought that a PCR'd fragment would work just as well, but tests show it to be only twice as good as chromosomal DNA (it should be at least 10-20 times better).
So I've been struggling to get good plasmid preps. I have two plasmids carrying this 9.4 kb novR fragment. One is the original clone, in an old natural ampicillin-resistant plasmid called pRSF0885. I have very old plasmid DNA preps, and some frozen cells from 1990 with the plasmid. The cells are still viable and resistant to ampicillin but my plasmid minipreps give no plasmid DNA. The old plasmid DNA efficiently transforms cells to AmpR, using a method that should produce plasmid transformants rather than replacement of the chromosomal allele, but again my plasmid preps give no or almost no plasmid DNA. I also have the same novR insert cloned into the CmR vector pSU2718 (almost identical to pSU20), but the plasmid yield is miniscule. So I'm beginning to suspect that something may be wrong with my plasmid prep method - I need to streak out something foolproof and prep it.
2. Tracking down the cause of non-reproducible colony counts:
Counting colonies is our most important research technique (yes, in some ways we're a very low tech lab). Normally the results are nicely reproducible, but lately I and the PhD student have been seeing bizarre discrepancies between replicate plates and between different volumes or dilutions of the same culture. After eliminating other variables (plates too dry? plates too wet? new brand of plates (nasty VWR plates with rounded edges?)? dirty spreading beads?) I did a test of 8 different combinations of different sources of BHI medium and of agar. This suggested that the problem may be with the old BHI agar we've been using (we were given two big buckets of it by a colleague). The good news is that this BHI agar is almost all gone, so the problem will solve itself. The bad news is that this BHI agar is almost gone, so we're going to have to start paying for more! (And I'm not confident that the problem is completely solved - there are still discrepancies, and tracking down the cause of irreproducibility is inevitably an exercise in frustration.)
What I should be doing:
3. Finishing up the honours student's Actinobacillus pleuropneumoniae experiment:
One of last year's honours undergrads left us with an almost-ready-to-submit manuscript lacking only some RNA-seq analysis and a remake and retest of one mutant. The other honours undergrad (now our summer student) has been working on the RNA-seq analysis, but we're still waiting for the final missing sequencing data. My job is to remake the mutant and test its competence phenotype.
The problem with her original mutant was that misannotation of a gene overlap had resulted in a mutant that deleted the last five amino acids of a gene that needed to be intact. Last month I spent ages designing primers to delete the correct segment, but the primers ahve jsut been sitting on my bench. I need to reconstitute them and (tomorrow) get instructions form the PhD student on where the PCR reagents are and how to use the PCR machine.
4. Learning R:
Our Life Sciences Centre R group is holding lots of workshops and help sessions, but by bad luck they've mostly been at times I can't attend. The summer student has given me some R homework, but it's buried on my desk.
5. Clean my desk:
To find the R homework.
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