2*CTAB extraction buffer, 110 mL (need to be freshly made, store for no more than a week)
Amount Chemical Final concentration
2.2 g CTAB powder 2%
11 mL 1M Tris-HCl (pH8.0) 100mM
4.4 mL 0.5M EDTA (pH 8.0) 20mM
30.8 mL 5M NaCl 1.4M
Add MQ H2O to 110 mL
[Note: Need to place CTAB buffer in 50ºC water bath or hot plate stirrer to get all the CTAB powder to dissolve]
Reagents needed
• Chloroform
• Isopropanol
• Ethanol
DNA Extraction
- Collect the plant materials (usually young leaves 50-100mg) in tubes with one metal ball in each tube, and put on ice to keep them fresh.
- Add 200 uL of “fresh buffer solution” (cold is OK) into each tube.
- Grind the leaves with Geno/Grinder ( 30s or 1min, then another 30s or 1min; the duration depends on the amount of tissue to be ground).
- Add another 200 ul CTAB buffer into each tube.
- Shake thoroughly and put them on the rack in the 65oC water bath for 1 hour. Invert the tubes several times to mix.
- Switch off water bath and take out the tubes. Put the tubes in freezer (-20C) to cool if still hot.
- Add 400 uL of Chloroform to each tube (Do it in fume hood!!!). Shake gently and continuously to mix (When chloroform is added and mixed, lids tend to pop up. Can open the lid to release the gas if necessary). Mix samples for 10 minutes at room temperature (Can do this manually or using the rotated mixer in the Molecular Lab).
- Centrifuge the tubes for 10 minutes at maximum speed (approx. 13,200 rpm) at room temperature.
- Transfer 200 uL of the clear upper phase into a fresh tube. Use yellow tips, and be careful not to disturb the middle layer of proteins.
- Add 200 uL of freezing cold isopropanol to each tube to precipitate DNA, and invert several times to mix.
- Incubate at room temperature for at least 10 minutes. Can be left overnight at 4C at this step.
- Centrifuge the tubes for 10 minutes at maximum speed.
- Gently pour off the supernatant (making sure the pellets are firmly attached to the bottom of the tube before doing this). Dry as much as possible with paper towel.
- Wash with 300 ul 70% cold ethanol and then centrifuge for 5-10 min at maximum speed (the duration of centrifuge depends on the number of the samples to be extracted). Pour off the supernatant. Dry the tube on an up-side-down position on a paper towel with the lids opened. Can leave at room temperature overnight to dry at this step.
- Alternatively, can put the tubes in an oven of no more than 55 C until dry (10 -15 minutes should be enough; Do not over dry them or otherwise they will become difficult to resuspend).
- Add 50 ul of 1*TE buffer to dissolve the pellets. Add 1ul RNase. Incubate at 37 C for 1 hour.
- Store at –20 C.
Stock solutions for DNA extraction (CTAB)
1M Tris-HCl (pH8.0)-1000 mL
• Dissolve 121.1 g Tris base [tris(hydroxymethyl)aminomethane] in 700 ml MQ H2O
• Adjust to pH8.0 with concentrated (32%) HCl (~42 ml)
• Mix and add MQ H2O to 1000 mL
• Sterilize by autoclaving and cool down in fume hood
• Label bottles and store at room temperature
[NOTE: The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be adjusted to the desired pH at the temperature at which they will be used. Because the pKaof Tris is 8.08, Tris should not be used as a buffer below pH ~7.2 or above pH ~9.0.]
0.5M EDTA (pH 8.0)-500 mL
• Dissolve 93.05 g Na2EDTA.2H2O in 350 ml MQ H2O
• Adjust pH to 8.0 with 10M NaOH
• Add MQ H2O to 500 mL
• Sterilize by autoclaving and cool down in fume hood
• Label bottles and store at room temperature.
10M NaOH-250 mL
• Make in fume hood.
• Dissolve 100 g NaOH in 150 ml MQ H2O (Become hot)
• Add MQ H2O to 250mL
• Cool down in fume hood
1xTE buffer-50 mL
• 0.5mL 1M Tris-HCl (pH 8.0)
• 0.1mL 0.5M EDTA (pH 8.0)
• 49.4mL MQ (Autoclaved) H2O
0.1xTE buffer-500 mL
• 0.5mL 1M Tris-HCl (pH 8.0)
• 0.1mL 0.5M EDTA (pH 8.0)
• 499.4mL MQ(Autoclaved) H2O
5M NaCl- 500mL
• Dissolve146 g NaCl in 350 ml MQ H2O
• Add MQ H2O to 500 mL