Troubleshooting
As this week comes to an end, several things have changed…for better or for worse.
The good news: My PCR modification succeeded in that the primers didn’t degrade before the transformations. I suspect that during the first few weeks, the annealing temperatures were too high, and since our primers had relatively low melting temps, they “died.” Also, the transformation into BL21 cells for protein overexpression worked as well, as I got confirmation from my protein gels. Now, those samples are purifying in the FPLC. Very Nice. So now, four out of the eight point-mutagenesis trials are working.
The other four…have problems. One mini-prepped DNA sample came back negative for point-mutagenesis, so I’m going to redo that sample. Another three plates didn’t have colonies, so I might redo the PCR for those primer samples and restart the whole procedure. If that doesn’t work, I’ll probably lower the PCR temps again. We’ll see.
On a side note, I’m really excited for the last few weeks of my fellowship, as it’ll be time to analyze all the data that I’ve gotten so far. And just today, I’ve received those DNA sequences for the mini-preps. Next week will be killer, as I’ll have to start on an abstract and poster design for the culmination of this program.