Tuesday, August 25, 2009

Now I'm trying to wean myself off experiments and do lab tasks only. The final thing I want to finish is growing up new(!) dpra::spec cells and putting them in the strain collection. I've saved up most of the lab tasks for tomorrow, when the new(!) workstudy student will be coming in to learn the ropes.

Friday, August 21, 2009

I collected the results from the transformation assay on the double mutant (RecJ-ExoI-), but the KW20 (dprA::spec) was, unfortunately, contaminated. I'll have to go back and check the aliquots next week.

Last presentation on Monday!!! :0

Thursday, August 20, 2009

I mostly tried to recollect what I had been doing before I'd left.

-I was cloning Hi comM, ligA, and recBD genes using pTOPO and transforming into Ec.
I wasn't getting any plasmids out of the clones, though, and SS didn't get any either, AND we ran out of pTOPO, so this project's benched for now.

-I had done a transformation assay on KW20 str-R cells (using MAP7 DNA).

-I was going to test the transformation frequency of KW20 cells containing a spec-R cassette in the dprA gene.
If transformation frequency was low, this test would confirm that the KW20 cells were indeed unable to express the dprA gene.

-I was also going to retest the transformation frequency of my RecJ-ExoI- double mutants.
I need to do more replicates to check my previous results (they had differed significantly from what Kumar et al. had reported).

So I did the latter two today.

Also, good luck to anyone who applied for the Lab Assistant position! I'd say the job requires responsibility, ability to keep yourself on task, and probably some degree of neatness would help, too. :)

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 H.in 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 H.in 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.

Wednesday, August 12, 2009

Restreaked KW20 (transformed with dprA::spec cassette).
Did a plasmid miniprep of strains (ligA cloning vector and comM cloning vector in E.coli) I had streaked out yesterday. Then I digested it with EcoR1 restriction endonuclease and ran the products on a gel.