Page 15 - Biology - XII

Materials Required
Plant material-Spinach (palak) leaves/green pea seeds (matar)/papaya (5–10 g), grinder, crushed dry ice,
CTAB (Cetyl Trimethyl Ammonium Bromide)-buff er solution (2.5%), chloroform: isoamylalcohol (24:1),
ice-cold isopropanol, 5 M NaCl, incubator/oven, water bath, and centrifuge

Procedure
1. Freeze the plant material overnight/use frozen plant material.
2. Grind the frozen material (3–5 g) with dry ice in a grinder or mortar and pestle.

3. Transfer the material into centrifuge tube and add 3 ml of CTAB buff er (CTAB buff er solution). Mix it
by inversion to form a slurry.
4. Incubate the material at 60°C for 30–60 min (in incubator or water bath).
5. Add 3 ml of chloroform: isoamyl alcohol (24:1) and mix the contents by inversion for about 10–15 min.
6. Centrifuge the contents at 8000 rpm for about 10 min.
7. Carefully transfer the supernatant liquid (containing DNA) to another 10 ml centrifuge tube.

8. Add 1.5 ml of 5 M NaCl and mix the contents properly.
9. Add ice-cold 0.6 volume of isopropanol. Let the content mixture stand at room temperature for
about 60 min.
10. This mixing will result in appearance of whitish strands which will fl oat in the solution. This is the
fi brous DNA extracted and can be removed/separated by spooling.
11. Record your observation in your practical record fi le.

Grinder
Beaker

Frozen green pea seeds Grinded material
+ Buff er solutions
(CTAB)

Frozen green pea seeds Grinding Centrifuge tube

At 8000 rpm for 10 minutes

Incubated Material (60°) +
3 ml chloroform: isoamyl alcohol

Centrifuge tube Centrifugation

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