2×CTAB extraction buffer instructions

2 × CTAB extraction buffer

● Product number and specifications:

Item number

name

specification

Storage

RTG2405-01

2×CTAB extraction buffer

200 ml

RT

RTG2405-02

reducing agent

1 ml

RT

Instruction manual

One

● Product introduction:

CTAB (hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide) is a cationic detergent with the property of precipitating nucleic acids from acidic polysaccharides from low ionic strength solutions. In high ionic strength solutions (>0.7 mol/L NaCl) CTAB forms complexes with proteins and polysaccharides without precipitating nucleic acids. The nucleic acid is separated by removing the impurities such as proteins, polysaccharides, phenols and the like by extraction with an organic solvent, and then adding ethanol to precipitate.

2×CTAB extraction buffer composition: 2% (w/v) CTAB, 100 mM Tris (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 0.2% (v/v) reducing agent (as needed).

● Storage conditions:

Store at room temperature for one year. Stored at 4 ° C after addition of reducing agent.

● Experimental steps:

1. 2× ready-to-use CTAB extract preparation:

The reducing agent was added at a final concentration of 0.2% (v/v), and 0.2 ml of reducing agent was added to 100 ml of 2 x CTAB extraction buffer. The 2X ready-to-use CTAB extract after addition of the reducing agent was stored at 4 °C.

2. Take 0.2-0.5 g of fresh plant material and grind into powder in liquid nitrogen.

3. Transfer the powder to a pre-cooled 1.5 ml centrifuge tube and immediately add 0.7 ml of 2 x ready-to-use CTAB extraction buffer, water bath at 65 ° C for 30 minutes, and mix intermittently.

4. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), gently invert the centrifuge tube and mix for 8-10 times, and centrifuge at 102,000 rpm for 10 minutes at room temperature.

5. Transfer the supernatant to another 1.5ml centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), invert the centrifuge tube for 8-10 times, centrifuge at room temperature 12000 rpm for 10 minutes. .

6. Transfer the upper aqueous phase to a new 1.5 ml centrifuge tube, add 0.6 volumes of ice-cold isopropanol, mix gently and ice for 30 minutes.

7. Centrifuge at 12000 rpm for 4 minutes at 4 °C.

8. Remove the supernatant, rinse the pellet with 1 ml of 70% ethanol, and dilute ethanol at 12000 rpm for 3 minutes at 4 °C.

At 9.4 ° C for 12000 rpm for 1 minute, the residual ethanol was drained with a pipette, and the precipitate was dried by a clean bench.

Note: The precipitate cannot be completely dried, otherwise it will not dissolve well.

10. Add 30-50 μl of ultrapure water or TE buffer to dissolve the DNA, and store at -20 °C until use.

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