Posted by: ericmjl | June 9, 2009

Late Night Labwork

Just as Calvin mentioned earlier, we have bacteria that love to grow, and bacteria that just hate to grow. Couple that with human error, and you have a recipe for late night benchwork.

Last Thursday, having rushed off to do some bike repair work after the Journal Club meeting, I forgot to prepare an overnight culture for doing the bacterial growth curve. So, on the next day, Calvin and I had to prep a day culture and stick it in the 30ºC shaking incubator. As was what we expected, we only started seeing growth at around 4PM, and that was just for the BW27783 strains. The DB3.1 and DH5alpha cells didn’t start growing until 7:00PM. What a wait – I went back and forth from my friend’s hot-pot dinner to make sure that I had dinner while trying to complete this single experiment!

But such late night labwork paid dividends in downstream processing for making competent cells. Because we had to grow up cells to and OD of 0.4 or so, we kept finding out that we hadn’t been able to time it right to reach that OD. Now, with the growth curve, as soon as the cells reach an OD between 0.1 and 0.4, we can use the growth curve to predict when the bacteria will hit and OD of 0.4 before chilling them on ice for the downstream steps.

In any case, with that out of the way, it’s a lot easier to do comp cells with less frustration, and we can now focus on assembly and transformation. More troubleshooting ahead, but I’m not too worried – things are progressing well now, and though it still feels slow, I think we’ve got about 90% of our groundwork settled (Tetracycline and Kanamycin construction plasmids are left) and our assembly of our E. coli machine can actually start rolling!

Alrighty, off to prep for the Journal Club meeting.

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