Biotech Bootcamp and Continuing Project

     At the start of this semester, we started with doing a "Biotech Bootcamp." This week of instruction included lab safety, lab protocol, as well as having time to teach/remind students how to pipette and make media/agar. One important protocol we talked about was regarding labeling. We clarified that all media, agar, bacteria, ect. needs to be labeled with the date, contents, initials, volume, concentration, type of bacteria (this is all depending on what the contents are). Labeling also was an issue when it came to waste beakers in the lab. Now all beakers that contain waste must be labeled clarifying whether it was chemical or bacterial waste. We also discussed lab cleanliness. This included washing your hands before and after doing anything in the lab and cleaning off surfaces. Something that was an issue last semester was people leaving powdered media on the scale after measuring, and not wiping things down property. All equipment must be cleaned before and after use to keep things sterile and clean. Our goal for this semester is to get papers out and to focus on deinococcus aquaticus and other deinococcus species. Finally, a media making class was taught to all of the students. We went over the protocol for making media and ensuring that we were all measuring all products the same.

    For the semester, I will be leading a small group in a project supervised by Chad. There are two sides of this project, but our group will only be working on one portion of it. This week, we completed a dilution series of the cDNA that was synthesized last semester from deinococcus radiosurans. We started with our stock cDNA and our primers for the gene SecA. This is a gene that we needed to verify the efficiency of so we could use it as a reference gene. We did a triplicate series of each sample for consistency. QPCR uses fluorescent light to see whenever the cDNA is duplicated. The measurements for our dilutions were:

45μL Nuclease Free H₂O

5μL cDNA

 

We then put the dilutions into a PCR plate. The total volume for the plate was 10μL, so we did the measurements below:

 

6μL Master Mix

4μL cDNA Dilution

 

Recipe we used for our Master Mix

    Once we made sure there were no bubbles and the plate was spun down, we put it into the PCR machine. The PCR conditions are listed below.

 

95 °C – 10s

60 °C – 60s

35 cycles

 

Our results told us that SecA is not a good reference gene to use for our project. Next week, we will inoculate D. rad onto a TGY plate, incubate and grow for 24 hours, then we will complete another round of RNA isolation so we can continue to search for a better reference gene.