On plates

Planting on Plates

Make MS plates: http://wahoo.nsm.umass.edu/content/ms-plates

  • To get weakling mutants started before potting in soil
  • Or to plant seeds on specified media
  • Or to see roots

Seed Preparation

  • Put appropriate number of seeds in a sterile microfuge tube
  • Sterilize with EtOH (or sterilize with bleach – see below)
  • Sterilize filter paper with EtOH, let dry in sterile hood (can set it on top of an open sterile petri plate cover
  • Add 95% EtOH to seeds for 5 min, dump seeds onto filter paper
  • let dry, tap onto agar
  • Make a line of seeds in agarose above the midline of the plate:
  • Tape plates with Micropore surgical tape
  • Refrigerate the plates for 2 days
  • Move plates to growth chamber
  • Stand plates on edge under the lights. Tape several together.

OR Sterilize with bleach solution

  • Add ~1mL of bleach solution to each tube
  • Cap and shake
  • Leave tube on its side for 20-30 min, shaking periodically
  • Put upright after last shake (so seeds settle)
  • In laminar flow hood
    • Use a pipetman that has been sprayed with ethanol and air-dried in the hood
    • Remove as much bleach solution as possible from the tube
    • Add ~1 mL sterile ddH2O, mix and let seeds settle again
    • Do at least 4 washes, to dilute out the bleach
    • Add ~1mL sterile 0.1% agarose (so seeds will be suspended in the liquid)

Bleach solution for sterilizing seeds

  • 7 mL sterile ddH2O (must be freshly made!)
  • 3 mL Chlorox
  • 1 uL Triton X-100 (detergent to help get bleach into seed crevices)

0.1% agarose for plating seeds

  • 100 mL sterile ddH2O
  • 0.1 g agarose . autoclave to melt; shake bottle after autoclaving