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):
- Place seeds in microcentrifuge tube.
- Wash with 4:1 ethanol to bleach solution. (I used 1ml of this mixture)
- Let sit for 10 min.
- Pipette out mixture.
- Wash twice with 100% ethanol, and discard ethanol.
- 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.
- 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).
- Add 0.22g per 50ml of water of MS media
- 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
- Add 0.2g agar (to make 1% gel)
- Heat to dissolve agar
- 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:
It’s made from the breadboard that has become my plant station, screws, and nuts. Pretty simple, and works amazingly well.