Spring Break….WOOT WOOT

Monday’s lab began with a detailed discussion into what constitutes an IJCM paper. Basically, the paper is a step-by-step process of building up your case of why a couple of your assigned strains are species based on comparison to previously described species. We will use this in an attempt to write an IJCM- worthy paper. Personally I knew they had rules on the format of their submissions, but I didn’t realize that they had so many of them.
Our first lab procedure of the day involved gathering our stock plates to be re-plated on to new media. After streaking the strains we parafilmed them and placed the plates in the incubator. The next task began with gathering the desiccation wells. We resuspended the cells by adding 500 microliters of liquid media (optimal media for each strain was used) and allowing them to sit for thirty minutes. We then swished the cells for thirty seconds each to mix up the wells contents. We then took ten microliters of each strain from the wells and spot plated each onto their optimal media. We spot plated six organisms per plate, parafilmed and placed them in the incubator.
After that we observed the results of the strains exposure to 9 kGy or radiation. Only strain twenty-seven grew, this strain is from the Sahara which makes it more likely to be a candidate for UV resistance due to possible link between desiccation resistance (which the organism obviously needs since it’s found in the desert). Our last task of the day was making PCR Master Mix and selecting our LRH-4 DNA extraction as our DNA (+) control.

PCR Master Mix (in order, 49.5 µl in each tube)
· X10 buffer 80 µl
· Geosp1 8.0 µl
· Geosp2 8.0 µl
· dNTPs 80 µl
· water 632 µl
· sample DNA 0.5 µl
· Taq pol. 0.5 µl
We hot-started the PCR mix this time in order to make sure the DNA is accessible to the polymerase. The Taq polymerase was added after the hot start because at those temperatures Taq polymerase becomes inactivated.
PCR Cycle: 40 cycles
· 98 C for 10 min (hot start)
· 90 C for 5 min (add Taq pol.)
· 93 C for 30 sec
· 51 C for 30 sec
· 72 C for 2 min
· 72 to 46 C for 10 min

The next lab day began with gathering contaminated carbon utilization plates to be plated onto new media. It is suspected that a contaminated tip contaminated the cell suspensions (as demonstrated by all of our strain 33 plates being contaminated) so we made new cell suspensions for each of our organisms by: adding 500 µl of liquid media, a loop full of your organism and vortexing the mixture multiple times. We spot plated 10 µl of each organism to new carbon source plates. After that we parafilmed them and placed them in the incubator.
The final task of the day was spot plating 10 µl of each of our strains on to new carbon utilization plates.
Carbon Sources:
· Control C
· Fructose FR
· Cellobiose CB
· Pyruvate PY
· Trehalose TR
· Sorbitol SB
· Myo-inositol MI
· Maltose MT
· Citrate CT
· Acetate AC
· Succinate SC
· Mannose MS
· Alanine AL
· Arginine AG
· Asparagine AS
· Histidine HS
· Lysine LY
· Threonine TH
We parafilmed and placed the new plates into the incubator. Sorry it took so long guys we had a lot of plates to do (labeling all of them made felt like it took forever) and making new cell suspensions took up quite a bit of time. Well I’ve be staying home for Spring Break, hopefully working on our paper. I hope everyone has a happy and safe break.