Lissa1: June14-June21

Wednesday, June 14

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

Thursday, June 15

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

Friday, June 16

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

Weekend, June 17-18

 * 1) Saturday
 * 2) 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)
 * 5) also other enzymes-get from VectorNTI
 * 6) Quantify gel data from AM