Wednesday, June 3, 2009

Instead of just saying "Today, I did another MIV...", I'll explain why I did it.

I have done this experiment 3 times with good results each time. I'm getting clean controls, ExoI- and RecJ- strains are transforming as well as (sometimes even better than) the wildtype, and the dprA- strain is transforming poorly (which goes with the hypothesis).

Here are some sample transformation frequencies for the RecJ- strain (just as an example):
For experiment E09: 4.1E-4
For experiment E10: 6.6E-3
For experiment E11: 7.6E-4

I'm leaving out a lot of information by not including other numbers, but just know that for each experiment, the wildtype's transformation frequency was in the same log (i.e. "E-4" or "E-3") as RecJ-'s. Also, RecJ-'s no DNA control (they weren't given MAP7) had very low transformation frequencies, like 1E-8 or 1E-9.

Sounds good? Well, the funny thing is that E09 used MIV-competent cells that were made on the same day as E10. (E11 used cells made at a later date.) This means the biological aspect of E09 and E10 were the same, and something in the technique caused 41 out of 100000 cells to transform the first time, then 660 out of 100000 cells to do so the second time around!

Also, I do 1 in 10 dilutions of the cultures when plating, but sometimes I don't get 1 in 10 numbers. For example, if I count 100 colonies on a 10^-1 plate, I should get approximately 10 on the 10^-2, then around 1-ish on the 10^-3, but sometimes I get things like 667, 8, then 3. Huh??? It's a head-scratcher.

Since I couldn't think of anything that I could have done differently the second time (I even rechecked the calculations), today I did another replicate. This one was with cells that were made on the same day as the E11 ones.

So look forward to more results tomorrow!

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