To do today

– Transforming my PSU20-Pdsred construct into Haemophilus. I did this yesterday but used the E.coli concentration of chloramphenicol and got no colonies.

– Count plates from yesterdays ComMExoI experiment. I scanned over the plates this morning and everything seems to be good, I’m just waiting until later in the day when the colonies will be larger and easier to count

–  Writing up a phage post. Phage recombination increases 100 fold in when cells are competent compared to early log. However competence induced phage recombination is low in both dprA and comM knock out mutant, which we can kinda explain due to their supposed roles of protecting DNA from degradation, but rec2 mutants(no translocation, should have zero impact on phage) also have low phage recombination rates when competent.

– Since I’m growing up KW20 and making plates maybe I could do a little side one-off experiment. I want to test whether persister cells, the ones not killed off by antibiotics have a different transformation frequency than the general population. I’ll probably wait on this as there’s some experimental design I need to do.  I’ll have to figure out what concentration of antibiotic I can use to kill off only a portion of cells(I prefer to use novobiocin since it doesn’t require an expression time). I’m already getting distracted by reading papers on the paradoxical effect of antibiotics when I should be doing phage research

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2 Comments (+add yours?)

  1. Rosie Redfield
    May 11, 2011 @ 03:30:32

    How did you do your Haemophilus transformation?

    Reply

  2. dnogas
    May 11, 2011 @ 10:06:38

    Grew KW20 until OD=0.187, MIV for 100 minutes, transfer 1ml cells into a test tube containing 40ul of plasmid, incubate for 30 minutes, afterwards added 400 ul of 80% glycerol, vortexed, let sit at room temperature for 10 minutes, added 5ml of dilution solution, mixed, and plated 100-400ul on 10 2ug/ml chloramphenicol plates

    Reply

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