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.

Tuesday, August 11, 2009

Sooo...
Yesterday's transformation of cloning vector pTOPO (carrying comM) into DH5-alpha was supposed to yield blue and white colonies, depending on whether the correct gene was inserted into the plasmid but...
I got very nice pearly white colonies (OK, not exactly pearly, but white nonetheless) for the comM and ligA vectors, but recBD didn't grow much at all, so I replated the recBD cells from yesterday (E.coli magic; you can't "replate yesterday's cells" with H.in).

There are, of course, a number of things that could have gone wrong:

1. Ligation didn't work properly.
I have the ligation products, so maybe I could run them on a gel after cutting with a restriction enzyme?

2. Transformation didn't work properly.
Try it again?

3. Plating didn't go over so well.
I've checked my calculations for ampicillin and X-gal.
Amp stock = 50mg/mL
X-gal stock = 40mg/mL
Want final amp concentration of 100ug/mL and final x-gal concentration of 80ug/mL.
So, to 50mLs LB agar, I added 100uL amp and 100 uL x-gal.
That works out...



Also transformed KW20 strain with dprA::spec construct that SS made last week. Later, I'll use this new dprA::spec strain to test the transformation frequency of the RecJ-ExoI-dprA- triple mutant.

Monday, August 10, 2009

Today I ligated Hin's comM gene into pTOPO cloning vector. Last week, I amplified comM through PCR and purified it (with a kit hehehehe). The ligation was very very quick; probably didn't even take 30 minutes! I also transformed this cloning vector into DH5-alpha (does blogger speak Greek?), a strain of E.coli, then plated the cells on LB again containing ampicillin and X-gal. It was ... funny working with something other than Hin (E.coli even smells different), but it's cool to actually do something that I've studied in class! I remember learning about X-gal and the colonies appearing white if the cloning vectors have ligated with a gene (blue if they haven't).

Thursday, August 6, 2009

Did PCR to amplify comM gene from KW20 x-somal DNA, then ran a gel to check the product. There were two bands, not one, so I increased the annealing temperature (from 50 to 55 degrees Celsius) and am running another PCR.

The plates from yesterday's dprA- survival curve were ready to count, but sadly, all of the plain plates got contaminated (turns out it was the BHI!!! gah!!). The kan plates were fine, and I did count them, but since the growth of dprA- Hin cells could have been affected by the contamination, I don't trust the numbers very much.

Tuesday, August 4, 2009

As RR suggested in this comment, I excluded the first two points of the curve because the cell were not growing exponentially at that point, or there were too few of them doing so to trust the measurements.

The new graph (above) shows that the dprA- growth curve has a slightly lower slope, rather than a drastically lower one.

Today I transformed KW20, RecJ-, ExoI-, and RecJ-ExoI- competent cells with MAP7 DNA and selected for novobiocin. Tomorrow, we'll see how well the double mutant transforms.

Also tomorrow, I'll be doing a dprA- survival curve and will also learn about the dprA::spec plasmid that SS made. I'll use this DNA to make triple mutants (RecJ-ExoI-dprA-!) and test their transformation frequencies.