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        Cetyltrimethylammonium bromide (CTAB) Pla

        互联网

        2235

        Procedure  

        1. Grind 2 to 5 g of frozen leaves to a very fine powder with a liquid nitrogen-cooled mortar and pestle.

        2. Add 25 ml of CTAB Buffer and transfer to a 50 ml tube.

        3. Incubate at 65℃ for 20 min with occasional vigorous shaking.

        4. Add 10 ml of Chloroform, shake well, and place on a tube inverter at room temperature for 20 min.

        5. Centrifuge at 1,000 X g for 5 min to resolve phases.

        6. Transfer the aqueous phase to a fresh tube, add 17 ml of Isopropanol, mix, and place on ice for 10 min.

        7. Centrifuge at 1,000 X g for 5 min to collect the precipitate.

        8. Discard the supernatant and dry the inside of the tube with a paper towel (do not dry the pellet).

        9. Add 4 ml of TE Buffer and dissolve the precipitate by gentle inversion.

        10. Add 4 ml of 4 M Lithium Acetate and incubate on ice for 20 min.

        11. Centrifuge at 1,000 X g for 10 min.

        12. Transfer the supernatant to a fresh tube, add 16 ml of 100% Ethanol, and incubate on ice for 20 min.

        13. Centrifuge at 1,000 X g for 5 min to collect the precipitate.

        14. Discard the supernatant and dry the inside of the tube with a paper towel (do not dry the pellet).

        15. Dissolve DNA in 900 μl of TE Buffer by gentle pipetting.

        16. Add 100 μl of 3 M Sodium Acetate. Divide the sample evenly into two microcentrifuge tubes.

        17. Add an equal volume of Phenol to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).

        18. Add an equal volume of Phenol:Chloroform to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).

        19. Add an equal volume of Chloroform to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).

        20. Add 2 volumes of 100% Ethanol to each tube and incubate on ice for 5 min.

        21. Centrifuge 5 min to collect the precipitate (approximately 5,000 X g).

        22. Discard the supernatant.

        23. Centrifuge for 5 additional min and remove as much of the remaining liquid as possible with a pipette. Do not dry the pellet.

        23. Add 100 to 250 μl TE Buffer. Dissolve the pellet by gentle pipetting.

        Solutions Phenol:Chloroform    1:1 Phenol:Chloroform
        4 M Lithium Acetate 3 M Sodium Acetate TE Buffer   
        1 mM EDTA, pH 8.0
         10 mM Tris
        CTAB Buffer   
        800 mM NaCl
        1% (v/v) Sarkosyl
        140 mM Sorbitol
        Autoclave
        22 mM EDTA
        220 mM Tris, pH 8.0
        0.8% (v/v) CTAB 


        BioReagents and Chemicals
        Cetyltrimethylammonium Bromide
        Sarkosyl
        Chloroform
        Phenol
        Sodium Acetate
        Ethanol
        Sodium Chloride
        EDTA
        Sorbitol
        Isopropanol
        Tris
        Lithium Acetate

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