S-STEM Scholar Malica Pend Sanchez Blog

 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...

  This week for the lab we continued to work with 8 tubes that have been overlapped and amplified on a gel we used the raming left in our PCR tubes which were a 15ul sample. So we used the 15ul of sample and 3ul dye and ran it on a 1.2% gel. The recipe for a 1.2% gel is 120ml TAE buffer and 1.44g of agarose.  In the image above you can see the ladder in our first well and then the third well is 1 which was running at 54 C and lastly, 8 ran at 34 C. from this image we were able to see 54 C which is the number run has a good pair of bands and the rest have little ghost bands.  So another day we decided since we were getting a good result on the high gradient at 54 C we will run another heat gradient but the temperature will be raised higher at 52 C-70 C to make it overlap. When we were making our 8 tubes with 8ul of tet, 5ul of left, and 11ul of the master mix which equals 24ul in each tube we have 8 of. sadly when we were making our tubes we ran out of left so we were only...

 In this week's lab, we believe we had a mix-up on tubes we were working with when doing a heat gradient of left and tet we ran our overlap tubes that should not have primers and run on a gel if we see something then we know our tubes got mixed up. So we ran the tubes we thought got mixed up on a 1.2% gel but a bigger one that needs 120uL TAE buffer and 1.44g of agarose. After it was run on a gel we saw nothing at all so were not exactly sure what even happened so we decided to start all over again. We worked on a gel cleanup. We used this kit for the gel cleanup. the instructions we followed were we cut out our DNA fragment from our agarose gel we made sure to trim the remaining excess gel. we then transferred that to a tube and weighed the gel slice. Then added 4 volumes of gel dissolving buffer to the gel slice. then incubate the sample between 37-55 C(typically 50C) inverting periodically until the gel slice is completely dissolved. Insert the column into the collection tu...

 This week for the lab we worked with our amplified sample of overlap. We made a 1.2% gel which is 3.6g of agarose and 30mL of TAE buffer. When we placed our product in the wells we used 10 ul of sample and 2ul of dye not using PCR water because we wanted a better view of what we had in our sample.  In this photo, you'll see in the second row our 6ul ladder and our fourth row is the 12ul sample in the gel. We ran that gel and it took the whole lab session so we worked on extracting plasma from Ecoli that was grown over the weekend. So we can have more tet since we already have a lot of left and right. We used the molecular biology Thermo scientific GeneJET Plasmid Miniprep kit. The website we used to follow the directions is thermofisher.com/studio first step was we resuspended the pelleted cells in 250ul of resuspension solution and pipetted up and down til we no longer had cell clumps. Then add 250ul of the lysis solution and 350ul of the neutralization solution, flipped ups...

 This week Monday started off with Friday's work and running our  Anna, Hilgarth, and lactonase sadly when we ran that through a gel we didn't get anything out of it. It was very unexpected to not see anything since our lactonase was our positive control and was 99% going to show up.  The first one is a ladder, space, Lactonase, anna 1, anna2, space, hilgarth 1, and lastly hilgarth 2 We decided to move on and amplify the left, tet, and right want to see if we could get a better vision of the final fusion. We had one test tube with undiluted primer and one with diluted primer in a gel since we want to see 2700 base pears. since we waited for that to run I worked on getting more experience and making more 1% gels for later lab sessions to be quick and ready then we stored those. The result of this amplifying failed again we had a band stuck in the well and a streak the reason that it could possibly be stuck is that we have a big piece of DNA that wasn't able to come out. We...