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!

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