- Get Ice and Liquid Nitrogen. 1 Liter Liquid Nitrogen is enough for approximately 40-80 samples.
- Prepare CTAB Buffer: For every 1ml of stock CTAB Buffer, put 5ul beta-mercaptoethanol and 0.2g PVP, stir until dissolved.
- Make sure the water bath is at 65C
- Flash freeze seed using Liquid Nitrogen (put seed in 1.5ml tube, then pour N2 in tube)
- Put seed in Ziploc plastic bag (cut the plastic bag ~3cmx3cm)
- Crush seed briefly using hammer from outside of the plastic bag (to avoid contamination)
- Put in 1.5ml tube, then flash freeze again
- Grind seed using small plastic grinder, wait until all N2 has evaporate
- Pour 650ul CTAB Buffer and mix, making sure the crushed seed dissolved completely (don't vortex).
- Put in ice while grinding other samples. Of course use new Ziploc plastic bag each time.
- After all samples are ground, put it in 65C Water Bath for 1.5 hour (Not more, or the DNA will be brown due to phenolics. Not less, or there will be less DNA.)
- Pour 700ul of 24:1 Chloroform:Isoamyl Alcohol to each tube (~0.5 volume) and mix.
- Centrifuge at 13000g for 15 minutes
- Prepare and label new 1.5ml tubes
- Carefully take the top layer as humanly possible into the new tubes. Do not get the bottom layer or the middle layer, as it will contaminate your DNA. With practice you will be able to get most of it.
- Put 600ml of Isopropanol into each sample (~0.5 volume) and mix gently (until it's all dissolved)
- Centrifuge at 6000g for 15 minutes
- Carefully decant supernatant. There should be a white pellet.
- Wash pellet with 70% Ethanol (Cold) (600ul, vortex 5s, then spin for 5 minutes)
- Carefully decant supernatant without disturbing pellet. Dry the pellet overnight or using a 55C bath for 10 minutes
- Dissolve the pellet in 50 μl TE, TrisHCl8, or water. TE is for long storage (years) or Tris-HCl pH8 for short time storage (months) or water for very short time (weeks). If you want to do straight PCR, water is the best since more salt will inhibit polymerase, but if you use TE, as an alternative you can make a dilution plate by diluting the DNA in water, that way the salt and other contamination is diluted.
- Quantify the DNA.
- Dilute the DNA as desired. Usually the final concentration is 10ng/ul.