New Characterization Project and Multimedia test, Ethanol Precipitation

    This week I have been joined by 5 new lab members as well as been given a second project. The project will be characterizing Deinococcus sonorensis as there are a number of on-going projects working on it, especially a very important bioinformatics project just on sonorensis being done by Lani. We will be looking at biochemical tests as well as some morphology ones. 

    The main split will be Madison and Carime working on the AMC project while Emme, Brian and Diana work on the characterization project. All of them will be kept up to date on the other projects and join when able, as well as make sure to see as many procedures as possible across both projects, but their main focus will be on one of the projects. 

    We started this week off with an ethanol precipitate while looking into and gathering media recipes for a multimedia test. The ethanol precipitate was performed on the six samples from last week with the hopes that it would lower their organic contaminate levels enough for qPCR. We also added glycoblue to easier see the pellet while performing the ethanol precipitate. 
    

Ethanol Precipitate

1. Dilute the sample to 200μl and add .1 vol 3M sodium acetate and 2.5 vol ice cold 100% ethanol before vortexing to mix thoroughly  

2. Precipitate overnight at -80°C

3. Centrifuge at full speed at 4°C for 15 minutes

4. Wash pellet twice with 0.5ml ice cold 75% ethanol, spinning at 4°C for 10 minutes each time

5. Remove ethanol then spin to remove any trace amounts of ethanol remaining.

6. Air dry pellet 10-15 minutes and resuspend in nuclease free water. 


    Unfortunately the ethanol precipitate did not work and we lost almost all of the RNA while the organic contaminates didn't change much. After reviewing the procedure the better choice would have been to precipitate at -20°C for 1 hour instead of -80°C overnight. 




    In terms of the sonorensis characterization, we started with a multimedia test. This involved making a number of various medias and inoculating them with sonorensis (and e.coli for a control) to see what nutrients they would be able to grow on. In total we made and inoculated TSA, NB, R2A, TGY, TGY salt at 1% 2% and 3%, Mueller Hinton, EMB, Mannitol, Maconcy, PEA and LB media. 












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