Field of Science

New RNA-seq work

(OK, I just checked, RNA-seq should be hyphenated.)

I've made a couple of posts about plans for new RNA-seq work on the Sense Strand blog: and

Now it's time to get down to work.

Here's the planned samples.  We have 26 on the list, but a standard run will only be 24, so two need to be dropped.  Conveniently, the two KW20-in-Trizol samples might not be needed, depending on the available small-RNA data for H. influenzae, so I won't consider those right now.

For the rest, we have 6 mutant strains.  Given the mixups that have occurred so far, it would be prudent to check these every way we can.

We are checking antibiotic resistances and will transform each strain when we grow its culture and collect the samples for the RNA preps. One of the Honours Zoology undergrads has already checked the toxin and antitoxin strains by PCR (she's the one who suffered most from the mixup), and we'll use the same primers to check the cultures we're sampling from.  We need the former RA's help to find the primers for the ∆hfq mutant, but luckily its phenotype is quite distinctive - in our whole collection I can only think of one other mutant that has its competence down 10-fold.  RR753's phenotype is also distinctive, hypercompetent, but not very.  The crp and sxy mutants have the same phenotypes (same drug resistance, same complete lack of competence.  We have lots of sxy PCR primers, but we'll have to check to see which ones will work with this insertion mutant.  And, for both crp and sxy there's the additional problem that miniTn10 insertions do not amplify well because of their end repeats.  I wonder if we have an internal primer for miniTn10kan.

The co-op tech has checked antibiotic resistances and frozen fresh stocks of all the strains.  She's inoculated the 4 strains for the MIV-competence preps, and tomorrow we'll toy to collect all those samples.  (No, I haven't done anything in preparation yet.)

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