Transformation Tuesday
We performed the yeast transformation of the bait and followed the procedure listed below. The yeast transformation was successful. After the yeast cells were transformed with the bait, we noticed that histidine was still present, so we decided to prepare the cells to be grown on media plates with 3AT. So far in the lab, I have made the media with five different 3AT concentrations. After the media is ready, I will transfer the yeast cells to the media. We will be looking for colonies that are able to produce higher levels of histidine.
Transformation Procedure:
For one transformation micro-centrifuge 500mL of competent cells at 500xg for 4
minutes.
Add 0.5mL in EZ 1 solution to wash the pellet. Vortex the cells and discard the supernatant.
Add 50uL of EZ 2 solution and vortex the pellet.
Mix 50uL of competent cells with 3uL of DNA (Bait) and add 500uL of EZ 3 solution and mix.
Incubate samples at 30˚C for 45 minutes.
Sterilize an L-shaped spreader by dipping in ethanol and lighting it on fire but be careful not to heat the spreader.
With an L-shape spreader, spread all the sample on yeast media plate that contains the –tryp/-Leu
Incubate the plate at 30˚C for 2-4 days so the transformed yeast cells can grow.
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