S-STEM Scholar Anh Nguyen's Blog

  Introduction      Since our failed plasmid extractions last week with our kit, GeneJET Mini Prep, we decided to switch to the ZymoPure Plasmid Kit instead. We hoped that the new kit procedure would help us achieve our desire concentration for this extraction. Our wanted concentration is 30 ul. Below is our procedure for the new ZymoPure Plasmid Kit that we spent this week doing.  Process     Before we start on the kit, we have to centrifuge a total of 3mL of culture in tubes A, B, Ae, and Be and incubate it in 500uL of multibuffer 2 times for 5 minutes. Then, we would incubate it in 600 uL of lysozyme for 45 minutes at room temperature. (Initially we did 30 minutes but it was not enough) After doing this process, we are able to move onto the kit. The kit has a different kind of protocol than the previous kit. For this kit, instead of starting with resuspension and supernatant, we used the ZymoPure P1, P2, and P3 individually and resuspending complete...

  Introduction:      Since our plates and flasks had plenty of growth and no contamination, we were able to move forward into plasmid extraction using the GeneJET Plasmid Miniprep kit.  Process:      Basically, we followed the protocol that was given to us from the kit. However, before starting the kit, we must extract the DNA of D. Aquaticus from the cells. We would use Eppendorf tubes to single out the bacteria pellet. We used 4 tubes at first with 1 mL of our samples from the 2 flasks, centrifuge it for 1 minute, and carefully pouring out the excess culture and avoid dropping the pellet (We do this for 3 times which is the same equivalent to 3 mL in total in each tubes). We would put 500 ul of multibuffer into each tubes and mix. Then, we incubate it for 5 minutes at room temperature them spin. We would repeat this another time. Finally, we are off to the kit.  Results:  Figure 1. 1% Gel Results Our extraction was too low and would no...

Introduction:      In lab this week, my group and I used the plates we've made and used a grown sample of D. Aquaticus  to inoculate into the plates.  Procedure:      Before we inoculate the plates, we had to prepare our  D. Aquaticus and its growth in order to transfer it. We got 2 flask with 40 mL of TGY Media and grew our bacteria in it. We then took gram stains of each flasks to see for any contamination but thankfully, it was am abundant of growth in our media. We decided to do 4 plates. First in our inoculation, we decided to burn the transfer loop before touching any culture/bacteria to avoid contamination. Then, we put the loop into the first 40mL flask and transfer it to 2 plates by streaking,  consisting heat fixing both the loop and the mouth of the flask each time when it comes in closely with bacteria or other media. We repeated this for the second 40mL flask but with the rest of the 2 plates. Lastly, once we were finished, ...