So the past two weeks have been pretty hectic, as I try to assemble some teaching labs for some basic biotech skills such as transformations and PCR, while simultaneously trying to attempt the multi-step synthesis of the first synthetic target for downstream biological testing. Waiting on chemical suppliers can be slow, but the arrival of our reducing agent and alcohol protecting group means we can move forward with the entire synthesis. Couple that with the arrival of the high pressure nitrogen tank for the 300Mhz NMR tomorrow, and things for once seem to be going ok in life.
You can see the successful transformation of DH5a super comp cells from NEB with pGREEN. The control plate is in the middle, surrounded by smaller LB-Amp plates with pGREEN+ transformants plated out at varying times of outgrowth. After addition of the SOC outgrowth media, I immediately plated 20uL of the cell suspension, and in other cases I let the cells sit in the media at 37C for 30 minutes, and 1 hour respectively, and plated 20uL. There did not seem to be any difference in transformation efficiency between the time points (though I know it’s impossible to tell transformation efficiency from those lawns). I didn’t bother with serial dilutions because I wasn’t concerned with transformation efficiency with pGREEN.
While I was waiting for everything I also brushed up on some gram staining skills, staining Staphylococcus aureus (little circle shaped bacteria) and E. Coli (the rod shaped bacteria).
Since Meldrum’s acid is an important reagent in the first step of the synthesis, it’s important to use as pure a product as can be reasonably obtained to insure high yielding 5′ acylation. As you can see from the picture on the left, shipped from Sigma it is granular with what looks like yellow/orange impurities. After dissolving in the minimum amount of hot acetone and allowing to cool, followed by an ice bath, nice long needle-like crystals start to form. The crystals are recovered via vacuum filtration and you’ll notice the comparable difference between the nice white crystals recovered and the residual yellow/orange waste sitting in the filtrate of acetone.
I managed to have a nice skype conversation with some prestigious scientists from the John Innes Center in the UK, so if this all fails, I guess it’s time for grad school. But, I’m staying positive, even though I’m poor right now.