Lissa1: July17-July26

Monday, July 17

1. Get rid of long-running SUMO gel -DONE
2. Pour new SUMO gel -DONE and store -DONE
3. Make new media SCD-trp+G418 -DONE
4. Pour new SCD-u-t plates -DONE
5. Make media for nocodazole arrests -DONE and refrigerate -DONE
6. Try to figure out unpolymerized acrylamide leftovers -DONE, 200 uL TEMED added

Tuesday, July 18

1. Do native extraction from blue-circle freezer yeast -DONE
2. Do phosphotase protcol -DONE
3. Load SUMO gel -DONE
4. Set up 3 overnights: ACYL379 (in YEPD), Fus3delta (in SCD-trp), Fus3delta/Kss1delta (SCD -trp + G418). -DONe

Wednesday, July 19

1. Arrive early! -DONE!
2. Pour 2 new small gels -DONE and store at 4 degrees -DONE
3. Grow up and flash freeze the samples for running the 2 small gels -DONE -(including pheromone arrests) -DONE
4. Set up 60 mL overnight of 403 in SCR-u-h. -DONE
5. Group meeting! -"DONE"

Thursday, July 20

1. Cut, transfer,-DONE set up overnight of pptase gel, pour new gel -DONE
2. Prep samples, -DONE load, and run -DONE the 2 small Westerns, and transfer, -DONEset up overnight
3. Nocodazole arrest! -DONE
4. Make a plate of the yeast ACL sent

Friday, July 21

AM:

1. Wash and image all 3 Western blots
2. Prep and load the nocodazole samples

PM: Lab Treat

Weekend, July 22-23

• Saturday: Lab Treat
• Sunday: Set up transfer + overnight of SUMO gel - BUFFERS HAD RUN OUT SO GEL WAS ONLY HALF-RUN. MADE NEW BUFFERS OF BOTH KINDS, REPLACED IN GEL APARATUS AND CONTINUED RUNNING.

Monday, July 24

1. Call our friend Jake (ext 122) and get some Luminol. -DONE, but no luminol
2. Learn how to use fluorescence microscope to look at induced cells
3. Learn exactly what is in ACL's strains
   1. Set up overnight of the appropriate strain with pGEV in it, and the parent strain
4. Learn how to do phenol DNA extraction
5. Cut, transfer, overnight incubation of noc. arrest gel, best done in PM -DONE
6. Start to set up model of PPL system on paper -DONE
7. Research ways to debug Pptase gel -DONE
   1. Add buffer to CIP reaction? -GOOD IDEA
   2. Increase amount of PPTase? -?
   3. Try another PPtase? -THIS SHOULD WORK
      1. Look at NEB catalog - Calbio has really nice phosphatases
      2. Look at other papers that have done Pptase -DONE

Tuesday, July 25

1. Wash and image Western blot -DONE
2. Phenol DNA extraction -DONE
3. Set up PCR of extracted DNA with the forward and reverse primers I ordered-DONE
   1. Also try integration verification primers from previous pGEV integration attempts. -DONE
4. ImageQuant -MOVED because people were using the computer by the typhoon
5. Transform SSY403GHC into pGEV strain and parent strain (starting from beginning of EZ trans)- by the time I got around to this part, my cultures had overgrown for a second time (they had overgrown while I was doing the Phenol DNA extraction) and I couldn't prepare the competent cells. I'll have to do this some other day.
6. Make freezer stock of Alejandro's strains. - equal parts yeast and glycerol, slow freeze -DONE
7. Size comparison of Kss1 and Fus3. Does it make sense to run them on a SUMO gel? -DONE, no