Rapid Method for DNA Extraction

Materials

          - Bacterial isolate cultured on an agar plate.

          - Inoculation loop

          - Incubator

          - Sterilized eppendorfs

          - Sterilized tips and pipettes

          - Centrifuge 

Solutions

      - Molecular biology grade water, autoclaved.

      - STE buffer (100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA, pH 8.0)

      - TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0)

      - Tris-saturated phenol (pH 8.0)

      - RNAse (10 mg/mL): Dissolve 100 mg of RNAse in 10 mL of sterile distilled water and incubate at 90°C for 10 minute to digest DNAse. Keep at -20°C in small fractions until use.

      - Chloroform

Procedure

        - A loopful of colonies from a cultured agar plate was resuspended in 400 µL of STE buffer and dissolved completely.

         - Cells were centrifuged at 8000g for 2 minute.

         - After removing the supernatant, cells were washed with 400 µL of STE buffer and centrifuged at 8000g for 2 minute.

         - Then, pellets were resuspended in 200 µL of TE buffer.

         - 100 µL of Tris-saturated phenol (pH 8.0) was added and vortexed for about 60 seconds.

      - The samples were subsequently centrifuged at 13000g for 5 minute at 4°C to separate the aqueous phase from the organic phase.

         - 160 µL of upper aqueous phase was transferred to a clean 1.5 mL tube.

       - 40 µL of TE buffer was added to make 200 µL and mixed with 100 µL of chloroform and centrifuged for 5 minute at 13000g at 4°C.

         - Lysate was purified by chloroform extraction until a white interface was no longer present; this procedure was repeated for three times.

          - 160 µL of upper aqueous phase was transferred to a clean 1.5 mL tube.

        - 40 µL of TE and 5 µL of RNAse (10 mg/mL) were added and incubated at 37°C for 15 minutes to digest RNA.

          - Then, 100 µL of chloroform was added to the tube, mixed well and centrifuged for 5 minute at 13000g at 4°C.

          - 150 µL of upper aqueous phase was transferred to a clean 1.5 mL tube.

          - The aqueous phase contained purified DNA and stored at -20°C.

          - The purity and yield of DNA was assessed spectrophotometrically by calculating the A260/A280 ratio and the A260 values to determine protein impurities and DNA concentration. 

Electrophoresis

Electrophoresis is applied to check the purity of DNA extraction. This method is useful for separation and analysis of macromolecules of DNA, RNA and proteins.