Lissa1: June14-June21

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Wednesday, June 14

  1. Grow up a culture of pGEV.
  2. SUMO
    1. Pour gel
      1. Fill stacking up to top
      2. Insert comb at angle
      3. If at first it doesn't succeed...
      4. More TEMED in running?
    2. Run gel
      1. Voltage = 500 (Note: load sample in center wells to mitigate smiling)
      2. Will it make a difference if we refresh buffer over time?
  3. Gal4 question?

Thursday, June 15

  1. Miniprep of pGEV
  2. Digest of pGEV.
    1. enzyme SnaBi?
  3. Overnight of yeast.
  4. Make plates?
  5. Check on SUMO gel?

Friday, June 16

  1. Yeast-pGEV Transformation
  2. Start Western. Overnight incubation w. antibody.

Weekend, June 17-18

  1. Saturday
    1. Wash and image.

Monday, June 19

  1. Take out transformations - NOTHING IS GROWING. BOOHOO.
  2. Grow up overnight culture - morning
  3. pour new sumo gel at 10% - afternoon
  4. digest pGEV - morning
  5. Sequence pGEV to see if it's what we thought it was - whenever server is up
  6. figure out how to get gradient gel for future Westerns - afternoon
  7. analyze ImageQuant data from Saturday's Western - afternoon
  1. use VectorNTI to make a model of pGEV - afternoon

Tuesday, June 20

  1. Re-do transformation the old way- no go. Overnight culture didn't grow. Streaked a new plate of 1030-1018 yeast to try again.
  2. do transformation with Alex's EZ kit - also couldn't do.
  3. start new sumo @400 V

Wednesday, June 21

  1. hope new sumo finishes - not done in AM. Made new cathode buffer and replaced all buffers.
  2. Do gel to see if PCR picked up an DNA

Thursday, June 22

  1. Set up transfer from gel to membrane and do overnight incubation on Western.

Friday, June 23

  1. Image Western
  2. Order BioRad gradient gel.
  3. Do old-school transformation.
  4. Do characteristic digest of pGEV (Acc1 - when it arrives)
    1. also other enzymes-get from VectorNTI
  5. Quantify gel data from AM