S-STEM Scholar Alex Hugenberg-Cox Blog

     I started this week with running a simple gel and extracting bands from it. We first ran a test gel to insure everything was as expected, and once that was confirmed we loaded the remainder of the sample into special double wide lanes to accommodate the increased volume. After running those gels, we cut out the DNA we wanted, and performed a get extraction. We used the gel extraction to remove any extra DNA, and isolate the specific DNA band we want.      We ran four samples, two of the left fragment and two of tet, aka L1, L2, T1, and T2.  the original confirmation gel    L1 L2 T1  T2   original tube mass (mg)  988.3 991.5  990.4  996.0   tube mass with gel (mg) 1323.8  1334.1  1247.0  1281.5   gel mass (mg)  335.5 342.6  256.6  285.5   amount of buffer needed (3x gel mass) ( μl)

  We started this week off with a get extraction. We first measured the peices of gel, combined it with four volumes of buffer, and nanodropped the readings.  Tet gel sample volume - 248.3 mg, 993.2  μl buffer, and nanodropped at 21.6  Left   gel sample volume - 192.9 mg, 771.6  μl buffer, and nanodropped at 10.1 We then used those two samples in overlap PCR, doing a heat gradient to help find our best annealing temperature. We diluted the tet sample with 16 μl tet and 48 μl PCR water. As we didn't get enough left sample from the gel, we also diluted an old left sample with 15 μl of left and 30 μl of PCR water. We also used an old right sample as we didn't get any form the gel extraction, and luckily enough we didn't need to dilute that.  We had eight tubes of left and tet, and one tube of left, tet and right just to try.  1 LT 54 °C   2 LT 52.3 °C   3 LT 49.7 °C   4 LT, LTR 46.3 °C   5 LT 41.7 °C   6 LT 37.7 °C   7 LT 35.3 °C   8 LT 34 °C           After running PCR and a