Isolation of Plant DNA using CTAB Protocol with a Silica Spin Column

CTAB buffers are widely used to isolate DNA from plants as they are effective for removing polysaccharides and polyphenol contaminants. Most CTAB protocols simply precipitate DNA which is followed by chloroform extraction. However, by incorporating a silica spin column into the protocol, higher purity DNA can be isolated. Silica spin columns have a glass fiber filter that captures DNA so that it can be more stringently washed. DNA is easily eluted with water of TE buffer. This added step results in cleaner DNA than extractions without silica spin columns.

Materials

• 200 mg leaf tissue
• CTAB Extraction Buffer (CEB 125-01, CEB 500-02)
• RNase A Solution (10 mg/ml in water, boiled to be DNase free)
• Chloroform/Isoamyl alcohol (24:1)
• Isopropanol (2-propanol)
• 70% ethanol (Ethyl alcohol)
• TE buffer (10 mm Tris, pH 8, 1 mM EDTA)
• Silica Spin Columns (SSC 100-01)

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CTAB Extraction Buffer
CEB 500-02, CEB 125-01

Available in 125 and 500 ml bottles.

Select Option 125 ml 500 ml

Silica Spin Columns
SSC 100-01

Available in bags of 100

Protocol

1. Using a mortar and pestle, cryogenically pulverize leaf tissue and add 200 mg to a 2 ml microcentrifuge tube.
2. Add CTAB Extraction Buffer (500 µl) mix, and then place in a water bath for 30 minutes at 60°C.
3. Once done remove the tubes and centrifuge for 10 minutes at 13,000 x g.
4. Transfer the supernatant to a new tube and add 5 µl of RNase A. Incubate the tubes at 37°C for 15 minutes.
5. Extract the lysate by adding equal volume of chloroform/isoamyl alcohol, vortex and then centrifuge.
6. Transfer the upper aqueous layer to a new microcentrifuge tube. Repeat this step until upper phase is clear.
7. Precipitate the DNA by adding 0.7 volume of isopropanol, and then incubate at -20°C for 15 minutes.
8. Transfer the supernatant to a Silica spin column and centrifuge for a 1 minute at 8,000 x g to bind the DNA to the column.
9. Wash the column twice with 250 µl 70% ethanol, centrifuging after each wash.
10. Elute the DNA in 20 µl to 100 µl of water or TE buffer.

Typical Results

The following graph is from corn. For comparison, the procedure was done in parallel without a spin column. The lower green line represents isolation without a spin column.

X

A spectrophotometric reading for an isolation of DNA from corn. For comparison, the procedure was done in parallel without a spin column. The lower green line represents isolation without a spin column.