S-STEM Scholars Sam Bennett's Blog

 Primers     We started off this week's research with a new project. We will be trying to create pigment-less Deinococcus (no orange). There are three sections we were focused on working with; left flank, right flank, and kanamycin resistance. To start the process we first needed to add primers to our solutions. We made a 20 micro-liter sample from 1 micro-liter forward primer, 1 micro-liter reverse primer, 2 micro-liter DNA sample, and 16 micro-liters of the master mix. After the solution was put together, it was placed inside the Thermal Cycler. The total process took over an hour and a half and went through 36 cycles total.       The 36 cycles consisted of 7 different steps. 1. 95 degrees celsius (3 minutes) 2. 95 degrees celsius (10 seconds) 3. 65 degrees celsius (30 seconds) 4. 72 degrees celsius (1 minute) 5. Goes back to step 2 (repeat 35 times) 6. 72 degrees celsius (7 minutes) 7. 4 degrees celsius (until we are ready to remove)     After ...

  Gel Electrophoresis     This week was a caveat from the previous weeks practice. The week prior we used practice gels to get our technique down for loading the wells. The practice gels were a lot more rigid compared to the actual gels used for the electrophoresis process. The basics of why we use gel electrophoresis is charging the molecules which then moves with the electric current through the gel, then verifying the base pairs within the sample used. We began by filling our chamber with 1x TAE as our buffer. For the samples we used 5 micro liters of DNA, 5 micro liters of water, and then 2 micro liters of loading dye. For the ladders we used 6 micro liters of the ladder DNA. Since this was also practice, a few of use shared gels. Once the gels were loaded with our samples and connected to our transformer, we began the process. For the transformer wee had it set to 85 mA, verified it was bubbling from both terminals, and waited for the samples to hit the back of the d...

 Gel Electrophoresis     This week we focused on making gels, as well as adding samples to the gels. The goal behind it was to get used to putting samples inside the gels since they can easily be penetrated if not careful. Gel electrophoresis is important in the lab setting for separating DNA, RNA, or proteins. It uses an electrical charge to pull the samples through the gel, and once your sample has ran through the gel, it is placed over a UV light and the bands are read. Compared with a "ladder", you can determine the size of your samples base pairs. We originally started with practice gels, since they are rubber it takes a lot more to puncture the surface. We added our ladders as well as samples to the practice gels.  This picture is the practice gel sitting inside of the the chamber. We have 2 "samples" in the middle, and 2 "ladders" on the outside. As you can see from the photos there is air bubbles inside the ladder wells (we do not want that).  ...