Arabidopsis Growth Try 4: Setup

I’ve got larger test tubes (1in diameter and about a ft in height), I’ve got plenty of water, and I’ve got a PhD. Looks like I’m ready to grow some plants! Here is the protocol ( adapted from Jan 22):

Cleaning the seeds (protocol provided by Pedro Nunes):

  1. Place seeds in microcentrifuge tube.
  2. Wash with 4:1 ethanol to bleach solution. (I used 1ml of this mixture)
  3. Let sit for 10 min.
  4. Pipette out mixture.
  5. Wash twice with 100% ethanol, and discard ethanol.
  6. Let the ethanol evaporate.

The seeds will sink to the bottom so it is fairly easy to pipette any liquid in the tube. After step 6 I’ll add some water so I can pipette the seeds into their growth media.

Preparing the growth media:

I growing seeds in 10 different mixtures of D2O/DDW: 0% D2O, 5% D2O, 10% D2O, 20% D2O, 30% D2O, 40% D2O, 50% D2O, 60% D2O, 70% D2O, and 80% D2O. I’m not doing a 99.9% D2O sample this time. Each sample will have 20ml of water total.

  1. I used 1 bottle of D2O (100g), 1 fresh bottle of DDW (100g), and one old bottle of DDW (~50g, from 1/22/13, stored in desiccator).
  2. Add 0.22g per 50ml of water of MS media
  3. Mix water in 20ml amounts in test tubes
    • 0% D2O – 0ml D2O, 20ml DDW
    • 5% D2O – 1ml D2O, 19ml DDW
    • 10% D2O – 2ml D2O, 18ml DDW
    • 20% D2O – 4ml D2O, 16ml DDW
    • 30% D2O – 6ml D2O, 14ml DDW
    • 40% D2O – 8ml D2O, 12ml DDW
    • 50% D2O – 10ml D2O, 10ml DDW
    • 60% D2O – 12ml D2O, 8ml DDW
    • 70% D2O – 14ml D2O, 6ml DDW
    • 80% D2O – 16ml D2O, 4ml DDW
  4. Add 0.2g agar (to make 1% gel)
  5. Heat to dissolve agar
  6. Allow to cool for gel to solidify

Planting the seeds:

Normally, I use a pipetter that one would use for small volumes to deposit the seeds, but because the tubes are so much bigger than the ones in the past I need to alter my method. Today I used a glass Pasteur pipet with a very nice long tip. I used the wrong kind of bulb, so it was more challenging than expected, but worked well enough.

Also because my test tubes are much bigger than before, I made the mistake of not purchasing a test tube holder. So I had to fashion one from spare lab components. Check out my system:

Test tube holder fr 25mm diameter tubes.
Test tube holder for 25mm diameter tubes.

It’s made from the breadboard that has become my plant station, screws, and nuts. Pretty simple, and works amazingly well.