MODIFIED CTAB METHOD for DNA EXTRACTION

           
            1. Grind young leaf tissues. Place about 50ul ground tissues into 2ml microtubes. Add 750 ul of 2x CTAB buffer and 50 ul of 20% SDS. Mix thoroughly.
                 Incubate at 65 oC for 30 minutes to 1 hour. Agitate every 15 minutes to homogenize buffer with ground tissues.

            2. Cool briefly and add 800 ul chloroform-isoamyl alcohol (24:1). Mix thoroughly. Spin at 10,000 rpm for 15 minutes.

            3. Carefully transfer the aqueous phase (top phase) into a new 1.5 ml tubes.

            4. Add 800 ul isopropanol, mix thoroughly and incubate at -20oC for 30 minutes or overnight.

            5. Spin at 10,000 rpm for 10 minutes. Decant isopropanol and wash pellet with 500 ul 70% ethanol, spin for 5 minutes, and then drain dry.

            6. Dissolve pellet in 100 ul TE + RNAse mix (100 ul TE: 1ul RNASE 10mg/ml) and incubate at 37oC for 30 minutes.

                (Optional)

            7. Add 10ul 3M NaoAc and 200ul absolute ethanol. Mix and precipitate for 3 hours or overnight.

            8. Centrifuge at 10,000 rpm for 10 minutes and decant supernatant.

            9. Add 200 µl of 70% Ethanol and spin for 10,000 rpm for 5 minutes. Decant supernatant and dry pellets.

            10. Add 50 µl TE and mix until the pellet is dissolved.


    DNA QUALITY CHECK


            1. Prepare 1% agarose (e.g. 2.5g agarose in 250ml 0.5x TBE).

            2. Dispense 2µl of homogenized DNA samples and 4µl of 2x BJ in each well of a re-used plate. Spin down.

            3. Dispense samples in the wells of the gel. Add 2µl of 50ng/µl Lambda at the end of the gel.

            4. Run the gel in 150V for 30 minutes.


    REAGENTS

  •     2X CTAB (1liter)
                20g CTAB dissolve in 860ml sterile ddH2O
                    Add: 81.82g NaCl
                               100 ml 1M Tris pH8.0
                               40 ml 0.5M EDTA pH 8.1
                               Autoclave and store at room temp

  • 20% Sodium Dodecyl Sulfate (SDS)
                    Dissolve 20 g SDS in 100ml of sterile nanopure H2O.
                    Do not autoclave.

  • 3M Sodium Acetate
                    Dissolve 123.1g NaOAc in 400ml of dsH2O.
                    Adjust the pH to 6.0 and volume up to 500ml

  • TE Buffer (250 ml)
                    1M Tris (pH 8.0) 2.5 ml
                    0.5 M EDTA (pH 8.0) 0.5 ml
                    Sterile nanopure water 247.0 ml