Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period3/Dailylog

5/27/13

KK, KP

We saw the results of our second electroporation today. Again, strains TT9901 and TT9907 took up the plasmid by electroporation, but the TT25281 transformation was again unsuccessful. We don't know what's wrong. Last time around I failed to push the cuvette entirely into the probes of the electrophorator. This time however, we know we followed the protocol correctly ...

In reference to strains TT9901 and TT9907, which were successful transformations from the first time, yesterday we swabbed these strains and grew them in overnight test tubes at 37 degress. The medium we used was LB Amp/Arabinose. We were hopeful that the following day (that's today) we would not see any turbidity, because the arabinose would have induced the expression of CRO, inducing Lambda to its lytic cycle. However, today we found disappointing results: our overnights were saturated with bacterial growth. This assay isn't of course the best way to test whether our system is working, but as it was intended to at least give us a ballpark gauge as to the functionality of our clone, we are concerned that something is not right.

5/29/13

-KP KK Today we plated TT9907 and TT9901. We plated them on LB arabinose/amp/x-gal plates. We plated different concentrations of TT9907 and TT9901 on each of the plates. We added 50, 100, and 300 microliters onto each plate. We will check the plates tomorrow hopefully to find plagues showing that arabinose triggered lysis of our lambda. We unfortunately didn't get any growth of our TT23821, so we streaked another plate from our original stab sample and we will try to re-electroporate TT23821 on Friday.

5/31/13

- Discussed results for 5.20 Mutagen Concentration Experiment -> will need to try this one more time with minor adjustment to experimental design: plate -4 dilution using x6 and x8 top agar.

- Discussed plans for next week.

- Made new LB and x6 top agar.

6/3/13

-KK KP Our PCR product was consistent with every other attempt to amplify CRO: it failed. Consistency is only a virtue if you're not a screw-up :) Anyway, our gel showed a faint band at the 300 base pair level, but too faint to be considered successful. We considered a list of reasons why our PCR reaction is failing and discussed them with Dr. Grose: We boiled our template DNA for 10 minutes, but Dr. Grose recommends 5 minutes We added 1 microliter of template; Dr. Grose likes to add 2 microliters. We did not use a master mix to combine our reagents with our template DNA. We added each reagent individually to each tube. Because we are pipetting such small volumes, there is a lot of room for error this way. The Phusion program we used was set to an annealing temperature that was 1 degree too low for Phusion. We did the PCR reaction one more time with Dr. Grose. Tomorrow we expect to see an enormous, fat, band!

6/5/13

Regarding our PCR attempt yesterday, we did see a thick fat band ... at the bottom of the gel! Once again our PCR failed. This has led us to propose several reasons why we can't PCR amplify CRO: 1) the Lambda prophage isn't actually in TT 9901 or TT9907, or 2) our primers aren’t functioning to amplify CRO. We streaked a few plates of TT9901 and TT9907 to subject to UV light. If we see plaques, then we will know Lambda has been induced. Meanwhile Dr. Grose is going to check that our primers are the correct ones.