1 DNA extraction - Cetyl trimethyl ammonium bromide (CTAB) method
 

 

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Equipment required


Weighing Balance

Pestle and mortar

Gloves

Forceps

50 ml polypropylene centrifuge tubes

Centrifuge

Micropipettes and tips

Temperature controlled water bath


Ice bucket

-20c freezer

4c refrigerator and/or cold room

Orbit shaker

Glassware

pH meter

Autoclave

 

It is advisable to sterilise all the glassware, centrifuge tubes, pipette tips, mortar and pestle before extraction to avoid possible contamination.

Materials and reagents


Sample of plant tissue (about 5g)

Liquid nitrogen (LN2)

Polyvinylpyrrolidone (PVP 360)

2x CTAB Extraction buffer pH 8.0 preheated to 65c

2-mercaptoethanol

Isopropanol (pre-chilled)

Tris EDTA (TE) buffer pH 8.0


RNase A (10 mg ml-1)

Chloroform: Isoamyl alcohol mixture (24:1)

3M Sodium acetate pH 5.0

70% ethanol

Absolute ethanol

Double distilled water (ddH2O)

Concentrated HCl

NaOH pellets

Composition of reaction mixtures

A.   2X CTAB Buffer (1 litre)

                 20 g of CTAB dissolved in 860 ml of ddH2O

                 81.82 g of NaCl

                 100 ml of 1M Tris pH 8.0

                 40 ml of 0.5M EDTA pH 8.0

                 Sterilise by autoclaving

B.  1 M Tris pH 8.0 (1 Litre)

                 121.1 g Tris

                 700 ml ddH2O

                 Dissolve Tris and bring to 900 ml

                 Adjust pH to 8.0 using concentrated HCl (about 50ml)

                 Makeup the volume to 1000 ml using ddH2O

                 Sterilise by autoclaving

C.  0.5M EDTA pH 8.0 (1 litre)

                 186.12 g of EDTA

                 750 ml of ddH2O

                 Add about 20 g of NaOH pellets

                 Slowly add more NaOH until the pH is 8.0

                 Make up the volume to 1000 ml using ddH2O

                 Sterilise by autoclaving

(Note: EDTA will not completely dissolve until the pH is around 8.0)

D.  TE buffer (1 litre)

                 10 ml of 1M Tris pH 8.0 (10 mM)

                 2 ml of 0.5M EDTA pH 8.0 (1 mM)

                 Make up the volume to 1000 ml.

                 Sterilise by autoclaving

E.  3M Sodium acetate, pH 5.2 (1 litre)

                 408.24 g of sodium acetate in 800g of ddH2O

                 Adjust the pH with glacial acetic acid

                 Make up the volume to 1000 ml

                 Sterilise by autoclaving

F.  Rnase A

                 Dissolve 10 mg of RNase in 1 ml TE buffer

                 Preheat the mixture to 80c for 10 minutes to inactivate DNases

G.  Chloroform: Isoamyl alcohol (500 ml)

                 480 ml of Chloroform

                 20 ml of Isoamyl alcohol

Procedure

1.  Grind 5g of leaves frozen with liquid nitrogen to a very fine powder in a cooled mortar and pestle. Wear protective gloves.  Liquid nitrogen (-196c) may cause cryoinjury on contact with bare skin.

2.   Add 1% polyvinylpyrollidone (PVP-360) to the buffer just before extraction, if high concentration of phenols is suspected

3.   Add 40l of 2-mercaptoethanol per 20ml of the buffer just before the use, preferably under a fume hood.

4.   Transfer the powder to a 50 ml tube. Add 20 ml of CTAB Buffer.

5.   Incubate at 65c for 30 min with occasional vigorous shaking.

6.   Add 20 ml of Chloroform: Isoamyl alcohol, shake well, and place on an orbit shaker at room temperature for 20 min.

7.   Centrifuge at 4,000 rpm for 30 min to resolve phases.

8.  Carefully pipette out and transfer the aqueous phase to a fresh tube, add 20 ml of pre-chilled Isopropanol, mix, and incubate at -20c or at 4c or on ice for 1 hour or overnight.

9.   If the nucleic acid precipitated is hookable, hook out the pellet to a new tube; else centrifuge at 3,000 rpm for 5 min to collect the precipitate.

10. Discard the supernatant and wash the pellet in 70% ethanol and then drain dry.

11. Add 5 ml of TE Buffer and dissolve the precipitate by gentle inversion.

12. Add 15 l of RNase A (10 mg/ml) and incubate at 37c for 30 minutes.

13. Add 0.5 ml of 3M Sodium acetate and 10 ml of absolute ethanol. Incubate the mixture at -20c for 1 hour or overnight.

14. If the DNA yield is good hook out the DNA to a new tube; else centrifuge at 3000 rpm for 5 minutes.

15. Rinse the pellets with 70% ethanol and drain dry.

16.  Dissolve the pellets in 500 l TE buffer; transfer the contents to a 1.5 ml microfuge tube.  Rinse the 50ml tubes with 200 l TE buffer. Add the wash to microfuge tube.

17. If any debris are noticed spin the microfuge tubes at 12000 rpm for 5 minutes. Transfer the DNA suspension to a clean, new tube.

18.  Store the suspension at -20c.