Friday, August 14, 2009

Today I did a plasmid miniprep on E.coli DH5-alpha cells that I had transformed with a cloning vector containing recBD genes. However, I must have done something wrong earlier, because when I ran the product on a gel, I didn't get any bands (I was expecting a supercoiled plasmid). So that was a little sad. I'll have to start back from the top, ligating genes into pTOPO then them into RbCl-competent E.coli DH5-alpha...

I also did a transformation assay on the KW20 strR cells that SS asked me to grow up and make competent earlier this week. Since it's friday (and I won't be in tomorrow), I'll have to ask RR to kindly take them out of the incubator for me.
Publish Post

Between experiments, I read a paper titled "A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient" by Palchevskiy and Finkel (2009). They also used exonuclease knockouts (RecJ and ExoI sound familiar?), but to study viability, not transformation. When exonucleases are not present, DNA is not degraded, so it can't be used as food to nourish the cell, so the cell (eventually) dies.

Lastly, I'm currently making competent the KW20 that was given dprA::spec DNA earlier this week. I'll check the transformation frequencies of this strain to check that they have indeed recombined dprA- DNA into the chromosomal DNA, then I'll probably extract this strain's chromosomal DNA and feed it to my double mutant RecJ-ExoI- cells in order to make a triple mutant.

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