Wednesday, June 14
- Grow up a culture of pGEV.
- Pour gel
- Fill stacking up to top
- Insert comb at angle
- If at first it doesn't succeed...
- More TEMED in running?
- Run gel
- Voltage = 500 (Note: load sample in center wells to mitigate smiling)
- Will it make a difference if we refresh buffer over time?
- Pour gel
- Gal4 question?
Thursday, June 15
- Miniprep of pGEV
- Digest of pGEV.
- enzyme SnaBi?
- Overnight of yeast.
- Make plates?
- Check on SUMO gel?
Friday, June 16
- Yeast-pGEV Transformation
- Start Western. Overnight incubation w. antibody.
Weekend, June 17-18
- Wash and image.
Monday, June 19
- Take out transformations - NOTHING IS GROWING. BOOHOO.
- Grow up overnight culture - morning
- pour new sumo gel at 10% - afternoon
- digest pGEV - morning
- Sequence pGEV to see if it's what we thought it was - whenever server is up
- figure out how to get gradient gel for future Westerns - afternoon
- analyze ImageQuant data from Saturday's Western - afternoon
- use VectorNTI to make a model of pGEV - afternoon
Tuesday, June 20
- Re-do transformation the old way- no go. Overnight culture didn't grow. Streaked a new plate of 1030-1018 yeast to try again.
- do transformation with Alex's EZ kit - also couldn't do.
- start new sumo @400 V
Wednesday, June 21
- hope new sumo finishes - not done in AM. Made new cathode buffer and replaced all buffers.
- Do gel to see if PCR picked up an DNA
Thursday, June 22
- Set up transfer from gel to membrane and do overnight incubation on Western.
Friday, June 23
- Image Western
- Order BioRad gradient gel.
- Do old-school transformation.
- Do characteristic digest of pGEV (Acc1 - when it arrives)
- also other enzymes-get from VectorNTI
- Quantify gel data from AM