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  Section: Biotechnology Methods » Tissue Culture Techniques
 
 
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Preparation of Plant DNA using

 
     
 
Content
Tissue Culture Techniques
  Tissue Culture Methods
  Plant Tissue Culture
  Plant Tissue Culture (Cont.)
  Many Dimensions of Plant Tissue Culture Research
  What is Plant Tissue Culture?
  Uses of Plant Tissue Culture
  Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance
  Demonstration of Tissue Culture for Teaching
  Preparation of Plant Tissue Culture Media
  Plant Tissue Culture Media
  Preparation of Protoplasts
  Protoplast Isolation, Culture, and Fusion
  Agrobacterium Culture and Agrobacterium — Mediated transformation
  Isolation of Chloroplasts from Spinach Leaves
  Preparation of Plant DNA using
  Suspension Culture and Production of Secondary Metabolites
  Protocols for Plant Tissue Culture
  Sterile Methods in Plant Tissue Culture
  Media for Plant Tissue Culture
  Safety in Plant Tissue Culture
  Preparation of Media for Animal Cell Culture
  Aseptic Technique
  Culture and Maintenance of Cell Lines
  Trypsinizing and Subculturing Cells from a Monolayer
  Cellular Biology Techniques
  In Vitro Methods
  Human Cell Culture Methods

Alternatively, the nonionic detergent cetyltrimethylammonium bromide (CTAB) is used to liberate and complex with total cellular nucleic acids. This general procedure has been used on a wide array of plant genera and tissue types.

Many modifications have been published to optimize yields from particular species. The protocol is relatively simple, fast, and easily scaled from milligrams to grams of tissue; it requires no cesium chloride density gradient centrifugation.

Additional Materials
  • CTAB extraction solution
  • 2% (w/v) CTAB
  • 100 mM Tris·Cl, pH 8.0
  • 20 mM EDTA, pH 8.0
  • 1.4 M NaCl
  • Store at room temperature (stable several years).
CTAB Precipitation Solution
  • 1% (w/v) CTAB
  • 50 mM Tris·Cl, pH 8.0
  • 10 mM EDTA, pH 8.0
  • Store at room temperature (stable several years).
Extraction Buffer
  • 100 mM Tris·Cl, pH 8.0
  • 100 mM EDTA, pH 8.0
  • 250 mM NaCl
  • 100 mg/mL proteinase K (add fresh before use)
  • Store indefinitely at room temperature without proteinase K.
High-salt TE Buffer
  • 10 mM Tris·Cl, pH 8.0
  • 0.1 mM EDTA, pH 8.0
  • 1 M NaCl
  • Store at room temperature (stable for several years).

CTAB/NaCl
Solution (10% CTAB in 0.7 M NaCl)

Dissolve 4.1 g NaCl in 80 mL water and slowly add 10 g CTAB (hexadecyltrimethyl ammonium bromide) while heating and stirring. If necessary, heat to 65°C to dissolve. Adjust final volume to 100 mL.
  • 2% (v/v) 2-mercaptoethanol (2-ME)
  • High-salt TE buffer
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 80% ethanol
Pulverizer/homogenizer: mortar and pestle, blender, Polytron (Brinkmann), or coffee grinder
  • Organic solvent-resistant test tube or beaker
  • 65°C water bath
  • Beckman JA-20 rotor or equivalent or microcentrifuge.

Procedure
  1. Add 2-ME to the required amount of CTAB extraction solution to produce a final concentration of 2% (v/v). Heat this solution and CTAB/NaCl solution to 65°C. Approximately 4 mL of 2-ME/CTAB extraction solution and 0.4 to 0.5 mL CTAB/NaCl solution are required for each gram of fresh leaf tissue. With lyophilized, dehydrated, or dry tissues such as seeds, 2-ME/CTAB extraction solution should be diluted 1:1 with sterile water. 2-ME should be used in a fume hood.
  2. Chill a pulverizer/homogenizer with liquid nitrogen (–196°C) or dry ice (–78°C). Pulverize plant tissue to a fine powder and transfer the frozen tissue to an organic solvent-resistant test tube or beaker. Use young tissue and avoid larger stems and veins to achieve the highest DNA yield with minimal polysaccharide contamination.
  3. Add warm 2-ME/CTAB extraction solution to the pulverized tissue and mix to wet thoroughly. Incubate 10 to 60 min at 65°C, with occasional mixing.
    A 60-min incubation results in larger DNA yields. If maximum yield is not important, 10 min should be adequate. If the tissue contains large amounts of phenolic compounds, 1% (v/v) polyvinylpyrrolidone (mol. wt. = 40000) may be added to absorb them.
  4. Extract the homogenate with an equal volume of 24:1 chloroform/octanol or chloroform/isoamyl alcohol. Mix well by inversion. Centrifuge 5 min at 7500 µg (8000 rpm in JA-20; ~10000 rpm in a microcentrifuge, for smaller samples), 4°C. Recover the top (aqueous) phase.
    Octanol, rather than isoamyl alcohol, is used because it may enhance isolation of nuclei. Slower centrifugation speeds are possible if centrifugation time is increased accordingly; a microcentrifuge may be used for small-scale preparations (150 mg starting tissue). After centrifugation, 2 phases should be evident with tissue debris at the interface.
  5. Add 1/10 vol 65°C CTAB/NaCl solution to the recovered aqueous phase and mix well by inversion.
  6. Extract with an equal volume of chloroform/octanol. Mix, centrifuge, and recover as in step 4 above.The aqueous phase may still be light yellow-brown.
Precipitate Nucleic Acids
  1. Add exactly 1 vol CTAB precipitation solution. Mix well by inversion. If precipitate is visible, proceed to step 8. If not, incubate mixture 30 min at 65°C.
  2. Centrifuge 5 min at 500 mg (2000 rpm in JA-20; ~2700 rpm in microcentrifuge), 4°C.
    Do not increase the speed or time of centrifugation, as the pellet may become very difficult to resuspend. If there is no pellet, add more CTAB precipitation solution (up to 1/10 the total volume). Incubate 1 hr to overnight at 37°C. Centrifuge 5 min at 500 µg, 4°C.
  3. Remove but do not discard the supernatant and resuspend pellet in highsalt TE buffer (0.5 to 1 mL per gram of starting material). If the pellet is difficult to resuspend, incubate 30 min at 65°C. Repeat until all or most of pellet is dissolved.
    Polysaccharide contamination may make it excessively difficult to resuspend the pellet. Read the A260 of the supernatant and discard the pellet if nucleic acids are present in the supernatant.
  4. Precipitate the nucleic acids by adding 0.6 vol isopropanol. Mix well and centrifuge 15 min at 7500 µg, 4°C.
    Ethanol can be used for the precipitation, but isopropanol may yield cleaner pellets.
  5. Wash the pellet with 80% ethanol, dry, and resuspend in a minimal volume of TE (0.1 to 0.5 mL per gram of starting material). Residual CTAB is soluble and is removed by the 80% ethanol wash. Further purification of the DNA with RNase A and proteinase K may be done using standard methods.
 
     
 
 
     




     
 
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