Kill Switch Week 38 (16th-22nd of September)

17th September 2019

1. Yeast colony PCR

Team members: Swenja & Ojas

Colony PCR was performed for 15 colonies of all three constructs.

Materials

15 colonies of the following plates: pTEF1-BAX+pGAL1-BI-1, pADH2-BAX+pGAL1-BI-1 & pGAl1-BAX (from old yeast transformation)

Procedure

20 µl of 20 mM NaOH was pipetted into the wells of a microtiter plate.
15 colonies of each plate were marked and transferred into the corresponding wells.
The plate was then put into a PCR machine and the samples 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 MangoMix.
9 µl of the master mix was added to 1 µl to the boiled samples.
The samples were put into a PCR machine with the following program: iGEM yeast transformation
1. 96 °C for 30 s
2. 96 °C for 20 s
3. 55 °C for 25 s
4. 72 °C for 1 min 30 s (1 min pr. kb)
5. 72 °C for 5 min
6. 12 °C infinite
7. Steps b. to d. have been repeated for 32 cycles

Results

As seen in the picture, there were no positive colonies for sample 2 (pADH2-BAX+pGAL1-BI-1). Opposed to that, all 16 colonies of sample 1 (pTEF1-BAX+pGAL1-BI-1) seem to be positive. For sample 3 (pGAL1-BAX) there was one positive colony, colony 10.

18th September 2019

1. Yeast colony PCR

Team members: Swenja

Colony PCR was repeated with 14 colonies of construct 2 (pADH2-BAX+pGAL1-BI-1). See the protocol on the 16th of September.

Results

Unfortunately, the gel images were all blurry. A possible reason for that could be an overheating of the gel chamber as the water was rather hot when the gel had been taken out. Please watch if something similar happens (white gel chamber on the left!). However, based on the pictures taken, it seems that all colonies were negative.

19th September 2019

1. Storing of the positive pGAL1-BAX culture

Team members: Iben, Swenja & Noël

Materials & procedure

The colony PCR from the 17th of September yielded one positive pGAL1-BAX culture. This culture was incubated at 30 ° C over night. The next day, the culture was plated on a Glu-U plate to preserve it. For long term storage a Glycerol stock (1 ml of culture + 250 µl of 50 % Glycerol solution) was prepared.