S-STEM Scholar Malica Pend Sanchez Blog

 This week for lab I first worked on a plasmid extraction  1.  At least 3 mL culture cells isolated. Do 1 mL of culture at a time. Spin down for a minute and pack pellet each time. 2. Wash w/ 500 uL multibuffers (×2). Mix by pipetting up and down. Let sit for 5 minutes. Spin down for a minute. 3. 600 L of 1mg/UL of lysozyme @ 37 °C for 15 minutes. Also pipette up and down to mix, no vortexing. DO NOT spin. 4. Then, add 100 L of 7X Lysis Buffer (Blue) and mix by inverting the tube 4-6 times. Proceed to the next step within 2 minutes. a. DO NOT spin. 5. Add 350 ML of cold Neutralization Buffer (Yellow and mix thoroughly. The sample should turn yellow when neutralization is complete. A precipitate will form. a. Invert an additional 2-3 times. Centrifuge for 2-4 minutes. Transfer supernatant (800 ML at a time) in spin column. Spin down for 1 minute and empty flow-through. a. May need to do multiple times. Add 200 ML of Endo-...

 This week in lab we ran a heat gradient on right and tet. in the vary being we genuily believed we were not going to get an overlap at all and then we did which was so exciting for us until we concerned maybe some mix up happen. So on Monday our heat gradient was 35C-60C we've been having problems with our right so were hoping we can see anything with overlap. Our 8 tubes ran 1-8 listing the heat gradient below: 1- 60C, 2-57.9C, 3-54.6C, 4-50.1C, 5-44.5C, 6-39.6C, 7-36.7C, 8-35.0C In each tubes we had 11uL Master Mix, 7uL PCR water, 2uL right, and 4uL tet = 24uL in each tube(8). Also made a big gel 1.2% recipe is 120mL Tae buffer and 1.44 agarose. Once the heat gradient was done we ran it on a gel and these are the results: first well is ladder, space, and then 60C through 35C we realized we really like 8(35C), 7(36.7C), and 2(57.9C) so we ran the remaining which was 45uL and then added 9uL od dye and ran that on a gel so we could do a gel extraction. we also ran a PCR overlap 35C...