Weekend June 24-25, 2006
- Maybe pour SUMO gel on Sunday night
Monday, June 26, 2006
- Brainstorm on why transformations are failing
- Make new freezer stock of analog-inhibited Cdc28
- Pour more SCD -U-T plates
- POur more SCD -U-H plates
- Use yeast freezer stocks to plate test transformations.
- Re-digest 404 with SnaBI and run an agarose gel to see if it worked.
- Grow up an overnight of pGEV e. coli so I can miniprep more tomorrow
- Pour a sumo gel with 12% acrylamide
- Order restriction enzymes from NEB, and run diagnostic digest on pGEV
- 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
- Run sumo gel without 379 alpha - and use peristaltic pump (make more buffer)
- Make new freezer stock of yeast from yesterday's overnight culture
- Transform SSY403
- Miniprep pGEV, pRS404 and pRS414 out of E. coli
- Do characteristic digests of pGEV and run on gel
Wednesday, June 28, 2006
- TALK AT GROUP MEETING TODAY
- Check on SUMO GEL
- FIX PERISTALTIC PUMP
- Make more anode buffer
- Learn to use pH meter
- Check on plates
- Order antibodies -on hold until I hear from Drew
- Anti-active MAPK
- What to do about anti-Cdc28 antibodies?
- When to do beta-estradiol stocks? Probably not until transformation is working.
- Think about pGEV issue/digest
- Do another pGEV digest, same as yesterday, just to be sure I've done it right.
- Run out old gel to get 'exact' length of EcoRV-cut fragment
- Grow new overnight of yeast?
- 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
- Do transfer and set up overnight incubation of Western
- Transform pGEV in to yeast, with 404, 414, and 416.
- Grow up overnight of 403 that are currently on a plate in the 30 degree room.
Friday, June 30, 2006
- Make new freezer stock of 403 chemically inhibited yeast.
- Start Western on overnight incubation
- Pour new SUMO gel
- Native extraction!