Attempt SDS Page and also mini prep to confirm that yeast transformations worked
Friday Nov 14th
Participants: Meenakshi, Lafia, Nikola, Natalie, Wesley, Johan
Objectives: Attempt SDS Page and also mini prep to confirm that yeast transformations worked.
SDS PAGE: After some discussion was decided not to do the SDS Page due to the following reasons: The gel we had was Tris-gel Native. Since it was not a denaturing gel, we would likely get smears that would be hard to distinguish. Also we did not have any casein protein to run a comparison.
Mini Prep:
Materials: 1 Zymo yeast mini prep kit 2 tubes of transformed bovine Alpha S1 2 tubes of transformed bovine Beta B 1 agarose gel electrophoresis with gelgreen rather than EtBr
Method (as per zymo recommended protocol, carried out in the hood which was made sterile):
1. Aliquot 1.5 ml of the yeast cells into 1.5 ml microfuge tubes and spin down the cells at 600 x g for 2 minutes. 2. Add 200 ul Solution 1 to each pellet.
3. Add 3 ul of Zymolyase™ to each tube. Resuspend the pellet by flicking with finger or mild vortexing.
4. Incubate at 37°C for 15-60 minutes (15 minutes is the minimal incubation time. Longer incubation is optional, but is suggested for stationary phase or older cells). 5. Add 200 ul Solution 2 to each tube. Mix well. 6. Add 400 ul Solution 3 to each tube. Mix well. 7. Centrifuge at maximum speed for 3 minutes. 8. Transfer the supernatant to the Zymo-Spin-I column. 9. Spin the Zymo-Spin I column for 30 seconds. 10. Discard the flow-through in the collection tube. Make sure the liquid does not touch the bottom part of the column. 11. Add 550 ul of Wash Buffer (ethanol added) onto the column with the collection tube and spin for 1-2 minutes. 12. Add 10 ul of water or TE and spin for 30 seconds-1 minute to elute plasmid off the column into a new 1.5 ml microfuge tube. For plasmids larger than > 15 Kb, incubate the column and elution buffer (water or TE) for 5 minutes before centrifugation to increase plasmid yields.
Ran gel electrophoresis on a 1% agarose gel for 40 minutes.
Result: Bands were extremely faint, inconclusive. Possibly around the 10 kb area.
Above miniprep was repeated on Nov 17th. to confirm result