Lissa1: June24-June30

Weekend June 24-25, 2006

1. Maybe pour SUMO gel on Sunday night

Monday, June 26, 2006

1. Brainstorm on why transformations are failing
2. Make new freezer stock of analog-inhibited Cdc28
3. DIAGNOSTICS:
   1. Pour more SCD -U-T plates
   2. POur more SCD -U-H plates
      1. Use yeast freezer stocks to plate test transformations.
   3. Re-digest 404 with SnaBI and run an agarose gel to see if it worked.
4. Grow up an overnight of pGEV e. coli so I can miniprep more tomorrow
5. Pour a sumo gel with 12% acrylamide
6. Order restriction enzymes from NEB, and run diagnostic digest on pGEV
7. Grow s288c (non-knockout yeast) on my old plates and see if they can survive at all - if they can, then the plates are ok - if they can't, then the plates are bad.

Tuesday, June 27, 2006

1. Run sumo gel without 379 alpha - and use peristaltic pump (make more buffer)
2. Make new freezer stock of yeast from yesterday's overnight culture
3. Transform SSY403
4. Miniprep pGEV, pRS404 and pRS414 out of E. coli
5. Do characteristic digests of pGEV and run on gel

Wednesday, June 28, 2006

1. TALK AT GROUP MEETING TODAY
2. Check on SUMO GEL
   1. FIX PERISTALTIC PUMP
   2. Make more anode buffer
      1. Learn to use pH meter
3. Check on plates
4. Order antibodies -on hold until I hear from Drew
   1. Anti-Fus3
   2. Anti-active MAPK
   3. What to do about anti-Cdc28 antibodies?
5. When to do beta-estradiol stocks? Probably not until transformation is working.
6. Think about pGEV issue/digest
   1. Do another pGEV digest, same as yesterday, just to be sure I've done it right.
   2. Run out old gel to get 'exact' length of EcoRV-cut fragment
7. Grow new overnight of yeast?
   1. If cells in -80 freezer are not competent, or if we decide the EZ transformation really doesn't work, we'll need new cells.

Thursday, June 29, 2006

1. Do transfer and set up overnight incubation of Western
2. Transform pGEV in to yeast, with 404, 414, and 416.
3. Grow up overnight of 403 that are currently on a plate in the 30 degree room.

Friday, June 30, 2006

1. Make new freezer stock of 403 chemically inhibited yeast.
2. Start Western on overnight incubation
3. Pour new SUMO gel
4. Native extraction!