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!

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