Lab 6: Mitosis

4 MatTeks per group of LL-GFP-tubulin

Double-strength inhibitors, made up in non-CO2 medium:

• STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

• need 1 mL aliquots 10 µM in non-CO2 medium for students

• Make 100 mM stock:

  • 1 g in bottle.
  • Mix with 27.5 mL DMSO
  • heat to 65C (~1 hr), vortex
  • if necessary, filter sterilize to remove insoluble particles.
  • save as 1 mL aliquots. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)

• Make 1 mM stock from the 100 mM stock

  • 10 µL 1mM stock
  • 990 µL DMSO
  • vortex
  • it will crystallize in the refrigerator. Warm and vortex to redissolve it.

• Make 17 mL 10 µM in medium for students

  • 170 µL 1 mM stock
  • medium to 17 mL
  • aliquot

• Make 10 µL aliquots of 1 mM stock

  • label says to add 990 µL medium to make 10 µM working solution

from Alyssa:madke STLC in DMSO and that she heated to 42C, and vortexed. She says it went from clear to yellow/brown. we use the reagent at micromolar - 1-10 µM. Her stock solution was 1mM.

students will be adding 1 mL of inhibitor to 1 mL medium in dish; the ready-to-use 1 mL (in medium) should be at least 2µM

• Taxol (20 µM final)
• soluble in DMSO, not water.
  • students pick either Taxol or nocodazole, so make only 10 each
  • each tube has 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)
  • add 490 µL to make 200µM in two tubes!
  • mix with medium in 15 mL conical to make 10 mL at 20 µM

• Nocodazole (3.3 µM final)

  • each tube has 3 µL 33mM
  • add 97 µL to tube to make 100 µL of 1 mM
  • 33 µL 1mM + 10 mL medium = 3.3 µM

• Nuc Blue from Invitrogen (or Fisher NC0291762), 1 bottle per station

  • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on