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)
    • students pick either Taxol or nocodazole, so make only 10 each
    • each tube has 10 µL 10 mM
    • 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