Friday, July 31, 2009

Grew up KW20, RecJ-, ExoI-, and RecJ-ExoI- cells in MIV to make them competent. Next week, I'll test the transformation frequencies of these strains.

I also counted colonies to determine the cfu/mL counts for the KW20, ExoI-, dprA-, ExoI-dprA- growth curves. I know this sounds trivial, but I get so happy when I check the incubator in the morning and the colonies look easy to count. By this I mean that they aren't bunched together on one side of the plate, they aren't only near the rim of the plate, there aren't too many bubbles in the agar, and that the colonies are nice and large (well, for Hin, anyways). Anyways, these plates were good to count.

Hm. More thinking will have to follow... but for now, happy long weekend! See you on Tuesday~

Thursday, July 30, 2009

Today's growth curve with KW20, ExoI-, dprA-, and ExoI-dprA- turned out pretty well in terms of OD values.

Here's the first graph. It's the OD values taken every hour, plotted on a log scale.

I fit exponential trendlines to each of the plots. When an exponential graph is plotted on a log scale, it'll turn out as a straight line. The slope of the line shows the growth rate of the culture; the larger the slope, the faster the cells are growing.
It really does look like the dprA- strain lags behind in growth, or maybe it dies easily overnight. Or maybe both.

Tomorrow, I'll see how the cfus turn out.

Wednesday, July 29, 2009

Today I ran a gel on the products of PCR on my kit-extracted RecJ-, ExoI-, and RecJ-ExoI- DNAs with ExoI primers. I have done this experiment before, but the last lane didn't show up, so I decided to do a replicate.
However, this time, the last two lanes didn't show up! Plus I got many more additional bands, the most noticeable one being around 700bp in length. It shows up in all lanes, so I'm guessing it could be the mastermix. Would it be worth adding the PCR ingredients to the individual reaction tubes, rather than making a mastermix? I've been using used buffer for my gels, maybe I should go with some fresh buffer next time. I hope my PCR skills aren't slowly fading away... :S I'll be repeating this experiment this week or early next week.

Tomorrow, I plan on doing a growth curve with these four strains: KW20, ExoI-, dprA-, ExoI-dprA-. I'll be measuring OD600 values and plating to determine the number of cfus (colony-forming units) in a mL. I'm doing this experiment to see how well the strains grow. If they don't grow very well, it might be a sign that the strain's mutation serves a (somewhat) vital function. Also, I've done this experiment with RecJ- and RecJ-dprA-, so the ExoI- set will be for completeness, too.

On Friday, I'll be making RecJ-ExoI- cells competent, as well as KW20, RecJ-, and ExoI- cells, too, since they'll be used as controls. Next week I want to use these cells to test their transformation frequency. Without two exonucleases, I'm expecting the transformation frequency to be high. Also next week, I plan to do a survival curve for the dprA- strain. This is to see how many cells will stay alive in MIV solution. It involves incubating dprA- cells in sBHI until they reach an OD600 of 0.2-0.225, then filtering and transferring them into MIV solution, and plating them (on plain sBHI plates? on kan plates?) at intervals (I'm thinking 0', 50', 100', and 150').

Also hoping the weather cools down next week!

Friday, July 24, 2009

On Thursday, I ran a PCR and a gel:
The top 5 lanes are the 1kbp ladder, the PCR products of MAP7, extracted RecJ-, extracted ExoI-, and extracted RecJ-ExoI- DNAs with ExoI primers. The bottom-right 5 lanes are the 1kbp ladder and x-somal DNAs of the same samples (so basically just the DNA, not a PCR). The bottom was to check that the x-somal DNAs had extracted nicely (which they had). The top lanes should look like this gel, which all of them, except the last, did. I don't know what's up with the last one. More on this later.

Today, I ran a gel on another PCR:
Lane 1: 1kbp ladder
Lane 2: MAP7 DNA + RecJ primers
Lane 3: kit-extracted RecJ- DNA + RecJ primers
Lane 4: kit-extracted ExoI- DNA + RecJ primers
Lane 5: kit-extracted RecJ-ExoI- DNA + RecJ primers
These guys should look like the gel in this post, which they do. I thought I didn't have any of the products of PCR with ExoI primers left, but I checked again, and I did! So I ran the PCR product of RecJ-ExoI- DNA + ExoI primers, the one that didn't show up last time. I was hoping last time had been an anomaly with the gel, but as you can (or can't, rather), see in the very last lane, nothing showed up again. I might have to repeat PCR with ExoI primers again.

Lastly, today I counted (:|) colonies from my transformation of KW20, ExoI-, dprA-, ExoI-dprA-, and pilA- strains with MAP7 DNA. Here are the transformation frequencies:

KW20: 3.8E-4
ExoI-: 4.5E-4
dprA-: 2.2E-7
ExoI-dprA-: 1.0E-7
pilA-: 1.2E-8

So the double mutant strain (ExoI-dprA-) can't transform very well. The pilA- strains were confirmed for SS. She mentions in this blog post that the strain is not naturally transformable.

Happy Friday!

Wednesday, July 22, 2009

I ran a gel on yesterday's PCR products, which were MAP7, RecJ-, ExoI-, and RecJ-ExoI- DNAs from the Oxford lab with ExoI primers.
The MAP7 and RecJ- lanes are aligned with the 2kbp rung. This shows that the ExoI gene in this samples is uninterrupted. The ExoI- lane is a little higher than the RecJ-ExoI- lane because the ExoI gene in the former is interrupted by a tetracycline resistance cassette, whereas the ExoI gene in the latter is interrupted by a kanamycin resistance cassette. The ladder's not too clear, but I'd estimate that the tet cassette is ~3kbp long and the kan cassette is ~1kbp long.

Well, now we know what the RecJ-, ExoI-, and RecJ-ExoI- DNAs are supposed to look like with RecJ and ExoI primers.

Today I also extracted DNAs from my RecJ-, ExoI-, and RecJ-ExoI- strains. I'll be running PCR on these to check that they look like the Oxford DNAs. The kit was a lot faster and easier to use than the manual extraction method. No phenol-chloroform, too.

Lastly, I counted colonies from a transformation I did yesterday with KW20, RecJ-, dprA-, and RecJ-dprA- strains and MAP7 DNA. The cells from this experiment are a different batch from my previous transformations, so I got a good biological replicate. This set also showed RecJ-dprA- cells are not great transformers.

Monday, July 20, 2009

I started off the day with some PCR. I ran RecJ primers with KW20, RecJ-, ExoI-, RecJ-ExoI- DNAs (the last three are from the Oxford lab). I did this experiment as a control. The results will show what we expect from the genotypes from the Oxford lab. Eventually I'll run PCR with the same types of DNA, but extracted from cells that I've transformed using the Oxford DNA.

It basically looks like this:

1. Oxford DNA => test with PCR
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v
2. I use it to transform wildtype cells into RecJ-, ExoI-, and RecJ-ExoI- cells
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v
3. I extract chromosomal DNA from these cells => test with PCR

I want to check that the DNA from step 3 is the same as in step 1.
The gel came out as expected. KW20 and ExoI- bands are lower because they're shorter; they're not interrupted by a tetracycline resistance cassette. RecJ- and RecJ-ExoI- bands are higher up on the gel because they're longer. The gene coding for RecJ has been interrupted by a tetracycline resistance cassette. They're quite faint, though. I suspect elongation time needs to be increased. So the gene for RecJ is about 2kbp long, and the tet cassette is about 2 or 3 kbp long (I'll have to check that somewhere).

Friday, July 17, 2009

Long live PCR!!!
Lane 1: 1kb DNA ladder
Lanes 2-4: SS's KW20 DNA with RecJ, ExoI, and Hi crp primers, in that order
Lanes 5-7: my KW20 DNA with RecJ, ExoI, and Hi crp primers, also in that order
I ran this gel for 45 minutes at 80V.

The bands are quite gloppy. Is it the new Taq?

The RecJ gene (HI_1214) is 1728 bp long, and the ExoI gene (HI_1377) is 1422 bp long. Both are interrupted by a tetracycline-resistance cassette (how long is it?). Hi crp is 675 bp long, so it's much smaller than what we expect for RecJ and ExoI.

I think the second band in the ExoI samples is due to annealing temperature (which was 45 degrees C for this reaction). SS also got a smaller second band in one of her PCRs, and she was going to lower the annealing temperature. The Tm (I forgot what the "m" stands for, but I think it means the temperature at which half of the primer is attached to the template DNA) of my ExoI_F primer is 57.2 degrees C, whereas my other primers (RecJ_F, RecJ_R, and ExoI_R) have Tms in the 47-50 range. So I guess the low annealing temperature made the ExoI_F primer bind with parts of the template that it was not specific to.

Happy weekend!

Thursday, July 16, 2009

Planning to make some more MIV-competent cells ASAP, as I've used them all in transformations. Actually, I did a transformation today. It's a replicate with KW20, ExoI-, dprA-, and ExoI-dprA- taking up MAP7 DNA and un/successfully expressing novobiocin resistance.

Also, we got new Taq today and I ran a PCR right alongside SS. If nothing shows up, woe is me. If I get bands, I'll be so relieved I might consider supporting Radio Paradise. (JK; that only came to mind because I hear it many, many times daily.) I'll put the samples in the fridge and run them on a gel tomorrow.

Tuesday, July 14, 2009

Through transformations with MAP7 DNA, it's starting to look like neither RecJ or ExoI are specifically blocked from degrading DNA by dprA's product.

Thursday, July 9, 2009

KW20, RecJ-, dprA-, and RecJdprA-- strains grew up quite nicely today. They were pretty much on par with each other in terms of OD, and tomorrow I'll see how the viable counts did.

I also counted colonies for the transformation of the four strains above with MAP7 DNA. Two transformations have shown that RecJdprA-- transforms quite poorly, comparable to the dprA- mutant.

Wednesday, July 8, 2009

I transformed KW20, RecJ-, dprA-, and RecJdprA-- strains with MAP7 DNA. I'm changing the concentrations that I plate, because last time, I got a lot of "film"s and "TMTC"s (too many to count).

Made KW20, ExoI-, dprA-, and ExoIdprA-- strains MIV-competent, which didn't take too long to grow up, thankfully.

It feels like today was a very busy day, and the sentences above probably don't do justice to how pooped I am from monitoring the MIV-competent cells, to diluting and plating the transforming cells, and getting stuff ready for a growth curve tomorrow. It's probably because I did it all simultaneously.

Monday, July 6, 2009

Today, to test how well RecJdprA-- mutants transform, I fed MAP7 DNA to KW20, RecJ-, dprA-, and RecJdprA-- strains. We already know how well the first three strains transform, but I'm doing them together so that I can compare across strains.

I also did a similar transformation with KW20 and pilA- strains for SS.

Lab meeting today was pretty cool, especially the stuff about "polonies". It really felt like JC was showing us a magic trick. George Church is my hero.

Sadly, it's raining outside and I think I'm coming down with a cold. :|

Friday, July 3, 2009

Above is a diagram I found to explain my last post. By "blob" I mean droplet B and by "lake" I mean droplet C. SS's samples resemble high surface tension droplet B, but my PCR products look like droplet C, which has low surface tension.

I wondered if:

1) PCR samples that show up on gels have high surface tension and samples that do not show up on gels have low surface tension. ==> No correlation. Although the MAP7 DNA + ExoI primer that SS ran did not show up on the gel, it was still a B droplet type.

2) The heat of the thermocycler had changed the surface tension. ==> Considering that I ran the PCRs on two different machines (ours and the Thompsons'), and SS and I both ran the same cycle, doesn't seem likely.

It seems SS and I are using different ingredients! :0

I have been mixing the PCR ingredients together (but not actually running PCR), but I've only been able to get droplet Cs.

Tune in next week for developments!
The PCR I ran yesterday was a replicate of one SS did last week. We used the same ingredients and thermocycler. Which means nothing's wrong with the stuff, but I'm using them improperly.

PCR variables and controls:

1. Used approximately 165ngs of lab stock MAP7 DNA. I've used this to transform competent cells before, so it shouldn't be weird. I could try some dprA::kan chromosomal DNA I have, but SS got a nice band with MAP7 last time.

2. Used Hi crp primers, which are SS's. She used them with MAP7 and got a product, so there's nothing wrong with the primers. Well, actually, maybe there is, since MAP7 and RecJ primers only showed up faintly and MAP7 and ExoI primers didn't show up at all when SS ran the PCR.

3. The gel electrophoresis equipment is fine, because the 1kb ladder runs nicely.

4. Is it the thermocycler, somehow? But the first time I tried this, I used the Thompsons', and that didn't work. Plus, as I've already said, SS used the same cycle on the same machine and got results.

5. Is there a specific way that I should be adding the ingredients? I always Taq last, but I don't have a certain order for the other ingredients. Should I be vortexing or centrifuging them? We don't have a lot of Taq left; maybe it's really old?

I've noticed that my products always seem to be very watery when I mix them with the gel dye. I can't explain this very well, but when I mix chromosomal DNA with the dye, the whole blob of liquid turns blue (and it remains a blob), but when I mix the PCR product with dye, the dye stays at the bottom and the blob turns into a flat lake, almost. I don't quite remember, but I ran the gel for SS's PCR, and her products seemed to remain a blob. I know that doesn't really confirm anything (yes, we know PCR's not working for you, Rhena).

Hm.

Thursday, July 2, 2009

Finally made the four strains MIV-competent today.

I still think the overnight method is faster, though, because you don't have the additional step of growing cells to OD600=0.1, which takes an additional day.

I also ran another PCR today, and not even the Good Luck Crystal could help. What is the problem? I added all the ingredients (I even checked them off as I went) in the correct amounts, and the cycler was set properly...

I found an online PCR troubleshooting guide. Hypothesis A ("Pilot Error") and I ("Bad Karma") look especially promising. Will read the page and try again later.