S-STEM Scholar Malica Pend Sanchez Blog

  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...