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Minimal Media Prep

Minimal Media Prep

  •  1000 µl pipette
  • 1 1000 mL beaker
  • 2 1000 mL graduated cylinders
  • 100 X MES (methyl-ethyl-sulfate)
  • pipette tips
  • 1000X KCl and CaCl2
  • RO Water (Reverse Osmosis)
  • 2 Spin Bars
  • pH Indicator
  • Kimwipes
  • BTP Buffer (bis-tris propane)
  • 2 storage bottles

1) Preparing 2L Minimal Media (1mM KCl, 1mM CaCl2, 5mM MES, 1% agar, pH=5.7 with BTP)

 1.1)  Obtain a 1000 µL pipette, and one 1000 mL beakers.
 1.2) Set the pipette to 1000 µL
 1.3) Add 20 mL 100x MES ((0.5M) methyl-ethyl-sulfate), solution to the 1000 mL beaker
 1.4) Dispose of tip in biohazard bag, place new tip on pipette, and add 2000 µL of 1000x KCl and CaCl2 solution to the same beaker. Dispose of tip in biohazard bag.
 1.5) Fill the beaker containing solution with RO water until it reaches the 800 mL mark.
 1.6) Sterilize a spin bar with 70 % EtOH solution and place in the beaker containing solution
 1.7)   Place the beaker on a stirring plate and set the dial to 7.
 1.8)   Check the pH of the solution with a pH indicator by removing the cover of the indicator, gently wiping with a Kimwipe®, and placing in solution.
 1.9) Turn up the stir dial until the solution moves in a circular motion.
 1.10)   To alter the pH of the solution, pipette BTP (bis-tris propane) buffer dropwise into the solution until pH reaches exactly 5.70. This should be done very slowly, as pH changes drastically with each drop.
 1.11) When pH reaches 5.70, remove indicator from solution, rinse with RO water, wipe with a Kimwipe®, and place in cleaning solution.
 1.12) Fix the spin bar to the bottom of the beaker by holding a larger bar on the outside of the beaker.
 1.13) Pour the solution into 1 1000 mL graduated cylinder. 
 1.14) Rinse the beaker that contained the solution with RO water, and pour in the graduated cylinders. (Do this at least 3 times)
 1.15) Fill the cylinders with RO water to the 1000 mL mark.
 1.16) Mix the solution by placing a sterilized spin bar in the graduated cylinder, then placing on a stir plate. When mixed, pour 500 mL of the solution into another 1000 mL graduated cylinder.
 1.17) Bring the volumes of both solutions to 1000 mL with RO water and mix as in 1.16.
 1.18) Pour solutions in labeled bottles, and set aside for future use.