Kill Switch Week 35 (26th of August-1st of September)

26th August 2019

Team members: Swenja & Noël

Colony PCR was performed for ten colonies of two constructs.

10 colonies of the following plates: Plate 3 22/8 ADH2-BAX1+GAL1-B1 & 22/8 Glu-U pGAl1-BAX1

20 µl of 20 mM NaOH was pipetted into PCR tube.
Samples of the chosen colonie were transferred into the PCR tubes.
The samples were boiled for 10 minutes at 99° C.
A mastermix comprising the following compounds was done (Volumes per samples): 2.5 µl miliQ water, 0.5 µl of each primer working solution (primers: YEA 95,94,85), 5 µl 2x primer solution). 5. 9 µl of the master mix was added to 1 µl to the boiled samples.
5. The PCR programm: iGEM yeast transformation was started.

Team members: Swenja

To confirm whether there are positive colonies among the yeast colony PCR samples, a gel was run.

To prepare the gel:
1. 1% agarose gel was taken from the common stock in electrophoresis room.
2. The Agarose-TAE buffer solution was poured into a labelled casting tray.
Setting up the electrophoresis chamber:
1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer (after 5 uses).
2. To prepare the sample to be loaded, 4 μL of each sample was added into a new PCR tube and mixed with 1 μL of loading dye.
3. The wells were loaded according to the pictures below.
4. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 15 mins.