#PhDaily Day 7 18/08/2015

Hi everyone!

An early (well earlier!) start for me today, in the NMR lab for 9.30 to again check on my samples. All looked well and we were good to carry on some further experiments, after the nitrogen fill. The NMR spectrometers are refilled with liquid nitrogen weekly, at which time no experiments can be carried out. Then to the NMR lab meeting at 10 am. No talk from students or staff this week, but a lengthy chat about the general upkeep of the labs, and a few people moving onto new jobs!

After the lab meeting I had to perform some maintenance on my protein sample for the NMR. Some precipitate had formed, as had a few small bubbles, both can have a negative effect on the NMR results. I remedied the sediment by using a hand-centrifuge (aka lethal-machine) to collect it as a thin layer at the bottom of the tube, where it won’t have any effect on the data. I then removed the bubbles by carefully manipulating the plunger that sits in the NMR tube. This is a fine art and today i did it for the first time unassisted! I popped my sample back in the NMR, and headed up to the microbiology lab at around 12 noon.

I had a bunch of FPLC protein purifications to do today, so i got started on the first, when i found out that my new bike was ready to pick up! I arranged for my awesome girlfriend to pick me up from work in my lunch break at about 1 pm, and go and collect my bike! I got back into uni around 2.20, headed up to microbiology to check on the FPLC (which had been equilibrating over my lunch break), then back down to NMR to set some more experiments up (which will end on Friday). Now 4.00 (how did it get to this time already?!). I then injected 3 consecutive samples onto the FPLC, taking a considerable amount of time…

It had got to 6.30, and now i had to try and determine the protein concentration in my FPLC outputs. This is done by a spectrophotemeter, and some small sums, which results in me knowing the protein concentration in mg/ml. This result was ridiculously high, and so completely unbelievable. I followed this with one repeat blanking the spectrophotemeter with the buffer solutions used (in case they were tricking the machine into thinking more protein was present), then doing a 1/10 dilution. Neither of these resolved the issue. After speaking to my supervisor, we decided to resolve it using a protein colour change assay, which did confirm the presence of protein. I will use this in the morning to determine the concentration of my samples. I left work at 7.45, very sleepy…

Back at work at 8 am tomorrow, to finish preparations for the big experiment! Tune in tomorrow to find out how it goes!

Thanks for reading,

Microbe Stew

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