Lissa1: August6-August14

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Contents

August 6

August 7

  1. Pour new SUMO gel -DONE
  2. Pour new small gels -DONE
  3. Set up overnights for the following experiments:
    1. Nocodazole arrest -DONE
    2. Induction experiment -DONE
    3. Make more unarrested 403 for phosphatase Western -DONE
  4. Make media for the nocodazole arrest. -DONE
  5. Plan EVERYTHING -CHECK
    1. Cloning
    2. Finalized experiments
    3. What to do about luminol/ECF deal? - OOPS, stil don't know
  6. Integrate new-found numbers/data in to model -DONE
    1. Maybe update model to include Michaelis-Menton kinetics -NOT YET, talk to Ty

August 8

  1. Do nocodazole arrest! -CELLS WON'T GROW:(
  2. Do induction experiment! -DONE
  3. Set up overnights for the following experiments: -MOVED UNTIL TOMORROW
    1. Fus3/Active Fus3 gels
    2. Phosphatase control
  4. PCR up pGEV out of ACLY700 -DONE
  5. Run a gel of the PCR -DONE
  6. If there's time, run a second gel and extract and purify the pGEV (which might not be pGEV, based on the sequencing data) -DONE

August 9

  1. PCR amplify the pGEV band I purified yesterday -DONE
  2. PCR out GEV insert from the original pGEV DNA -DONE
  3. Run gels of these PCRs and do gel extraction -DONE
  4. Digest pGEV - no time
  5. Setup overnight of pRS-405 in DH5alpha, in LB + Amp -DONE
  6. Streak new plate -DONE
  7. Setup overnight of samples for Fus3, etc -DONE
  • Unfortunately, I've got a cold and need to rest:(

August 10

  1. Prep Fus3 samples (all the way to sample buffer) and store -DOne
  2. Get concentrations on all DNA - DONE
  3. Miniprep 405 out of E. coli -DONE
  4. Streak out new 403 plate from freezer stock -DONE
  5. Digest pGEV, run on gel to check -DONE
  6. Design diagnostics for prs405 DNA -DONE
  7. Digest 405 -DONE
  8. Do PCR cleanup - DONE

August 11

  1. Make new SCR-u-h media!
  2. Do native extraction and phosphatase experiment on Noc. arrested yeast
  3. Load SUMO gel and start run
  4. PCR up GEV insert out of miniprepped pGEV DNA

August 12

  1. Gel-extract and purify all digests/PCRs. -DONE
  2. Check on SUMO gel l- SOMETHING IS VERY WRONG. CHANGED ALL BUFFERS, CHECKED ALL CONNECTIONS, SET UP AGAIN. DID NOT SEE BUBBLES AT ALL. 1 HR LATER IT HAD MOVED A LITTLE BIT....

August 13

  1. The plan was to set up the transfer of my gel today, but whatever problem was present yesterday is also present today. I think a wire must have snapped inside the gel box? It's giving 400 V but only 2 mA of current...

August 13, 2006

  1. Digest my new PCR-ed fragments that hopefully are pGEV with the double digest -DONE
  2. At the end of the day, do the ligation
  3. Think about this week's very complicated schedule -DONE
  4. Set up overnight of my newly-streaked-out 403 cells to do noc. arrest and also to set up new samples for phosphatase control -DONE
  5. Get opinions on induction experiment -DONE
  6. Set up new overnight for another induction
  7. Scrap old SUMO gel and pour a new one
  8. Prep and run 2 new small Fus3/etc gels -DONE
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