(I'll add some explanations later.)
1. Mutagenize more RR805 DNA, using a range of high EMS doses (10, 15, 20, 25, 30, 40 min in 50 mM). Transform this DNA directly into competent KW20 (without EMS inactivation or DNA purification) and select for CmR and maybe for NovR.
2. Mutagenize RR805 cells, using a range of high EMS doses (from expt. #180, 80 mM for 1 hr gives ~10^-2 survival). The cells don't need to survive, because I'll just grow the culture for a couple of hours and then extract all the DNA and use that DNA to transform KW20 to CmR.
For both 1 and 2, then pool CmR transformants and transform at low cell density to StrR with RR514 DNA. Test individual StrR colonies for hypercompetence by colony transformation with MAP7 DNA.
3. Mutagenize NovR and NovS PCR fragments (made by the sabbatical visitor), using the same EMS concentrations as in experiment 1. Then test the effects of the EMS mutagenesis by transforming each DNA into KW20, looking for gain of NovR in cells transformed with the NovS DNA, and loss of transforming ability of the NovR DNA.
I can do experiments 1 and 3 today (if I first pour lots of plates). I can then do Experiment 2 tomorrow or on the weekend, once the cells have grown up.
1. I must have put too little chloramphenicol in the Cm plates for this experiment, because all the cells grew on the Cm plates. I need to repeat this experiment.
3. Increasing exposure to EMS caused decreased transformation by the NovR fragment, as it should, but the corresponding exposures of the NovS fragment gave no NovR transformants, indicating no detectable mutagenesis. So the decrease seen with the NovR fragment may just be due to damage, not mutation.
2. My streak of RR805 cells has grown nice little colonies.
I've inoculated one of the RR805 colonies for an overnight culture, so I will be able to do the experiment 3 cell mutagenesis tomorrow. And tomorrow I'll make lots and lots of Cm plates, with the right amount of chloramphenicol, so I can also repeat experiment 1.
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