Comments
Genomic DNA extracted from environmental samples, e.g. soil and sediment, contains
inhibitory substances for PCR. Purifification of genomic DNA with CTAB improves PCR
amplication. Basically, genomic DNA is extracted from soil with any protocols (e.g. FastDNA
SPIN Kit For Soil from MP Biomedicals,USA), then purified with CTAB method. The
concentration of genomic DNA is quantified with Nanodrop or Qubit assay. Finally, dilute
DNA concentration to 10 ng/µL and store at -20oC for future application (Reference: Mao et
al., Environ Microbiol. 2013 15:928-42).
DNA purification with CTAB
1. Pre-warm CTAB (Cetyltrimethylammonium Bromide) working solution (10% CTAB/0.7
M NaCl) to 65oC in a 1.5 ml tube in the heat block. Meanwhile, proceed to step 2 and 3.
2. Transfer ~100 µL of extracted DNA into 1.5 mL tube. NOTE: If DNA volume is not 100 µL,
adjust volumes of NaCl and CTAB solution accordingly.
3. Add 16.5 µL of 5 M autoclaved NaCl solution to the tube containing 100 µL DNA.
4. Add 12 µL of warm CTAB working solution. Mix thoroughly and incubate at 65 oC for
15 minutes.
5. Add 128 µL of chloroform: isoamyl alcohol (24:1). Mix carefully but thoroughly.
Centrifuge at maximum speed (14,000 x g) for 5 minutes.
6. Carefully remove aqueous phase ( top layer) to a clean and labeled 1.5 ml tube (about
125 µL). Add 250 µL of cold 100% EtOH to precipitate DNA. Mix thoroughly and incubate
the tube on ice or in a -20 oC for 2 hours or overnight. Centrifuge at maximum speed for
15 minutes.
7. Discard the supernatant. Add 125 µL 70% cold EtOH, flick to mix, then centrifuge at
maximum speed for 5 minutes. Repeat this step one time.
8. Remove supernatant. Air dry the pellet for about 30 minutes.
9. Resuspend pellet in 100 µL ddH2O.
10. Measure the DNA concentration with Qubit Fluorometer (Invitrogen) or Nanodrop.
Dilute DNA concentration to 10 ng/µL and store at -20oC.