R-DNA Technology

Experiment 1

Aim of the Experiment:

Plant DNA Extraction by CTAB method.

Introduction:

Plant materials are among the most difficult for high quality DNA extractions. The key is to properly prepare the tissues for extraction. In most cases this involves the use of liquid nitrogen flash freezing followed by grinding the frozen tissue with a mortar and pestle. Liquid nitrogen is difficult to handle and it is dangerous in an open laboratory environment such as a classroom. For this reason, we have modified a very simple plant DNA extraction protocol to use fresh tissue. We have also used tissue prepared in advance by dessication.

Principle:

Isolating DNA from plant tissues can be very challenging as the biochemistry between divergent plant species can be extreme. Unlike animal tissues where the same tissue type from different species usually have similar characteristics, plants can have variable levels of metabolites and structural biomolecules. Polysaccharides and polyphenols are two classes of plant biomolecules that vary widely between species and are very problematic when isolating DNA. Contaminating polysaccharides and polyphenols can interfere with manipulations of DNA following isolation.

Methods are available that effectively remove polysaccharides and polyphenols from plant DNA preparations. The use of CTAB (cetyl trimethylammonium bromide), a cationic detergent, facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, can aid in removing polyphenols. CTAB based extraction buffers are widely used when purifying DNA from plant tissues.

One option for purifying DNA using CTAB exploits that polysaccharides and DNA have different solubilities in CTAB depending on the concentration of sodium chloride. At higher salt concentrations, polysaccharides are insoluble, while at lower concentrations DNA is insoluble. Consequently, by adjusting salt concentration in lysates containing CTAB, polysaccharides and DNA can be differentially precipitated.

Polyphenols are compounds that contain more than one phenolic ring (e.g., tannin), a structure that binds very efficiently to DNA. They are naturally occurring in plants, but are also generated when plants have tissue damage (browning). Upon the homogenization of plant tissues, polyphenols are synthesized by liberated polyphenol oxidase. The addition of polyvinyl pyrrolidone prevents the interaction of DNA and phenolic rings by binding up the polyphenols.

Materials required

1. CTAB buffer: 2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone,100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, or CTAB Extraction Buffer

2. Centrifuge (up to 14,000 x g)

3. Isopropanol

4. 70% Ethanol

5. 2 ml centrifuge tubes

6. TE Buffer (10 mM Tris, pH 8, 1 mM EDTA).

Procedure:

1. Plant samples can be prepared by cryogenically grinding tissue in a mortar and pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room temperature. In either case, a fine powder is best for extracting DNA.

2. For each 100 mg homogenized tissue use 500 µl of CTAB Extraction Buffer. Mix and thoroughly vortex. Transfer the homogenate to a 60°C bath for 30 minutes.

3. Following the incubation period, centrifuge the homogenate for 5 minutes. at 14,000 x g.

4. Transfer supernatant to a new tube. Add 5 µl of RNase solution A and incubate at 32°C for 20 minutes

5. Add an equal volume of chloroform/isoamyl alcohol (24:1). Vortex for 5 seconds then centrifuge the sample for 1 min. at 14,000 x g to separate the phases. Transfer the aqueous upper phase to a new tube. Repeat this extraction until the upper phase is clear.

6. Transfer the upper aqueous phase to a new tube. Precipitate the DNA by adding 0.7 volume cold isopropanol and incubate at -20°C for 15 minutes.

7. Centrifuge the sample at 14,000 x g for 10 minutes. Decant the supernatant without disturbing the pellet and subsequently wash with 500 µl ice cold 70% ethanol. Decant the ethanol. Remove residual ethanol by drying in a SpeedVac.

8. Dry the pellet long enough to remove alcohol, but without completely drying the DNA. Dissolve DNA in 20 µl TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet may need warming in order to dissolve.

9. Run the isolated DNA in agarose gel electrophoresis.

• Agarose Gel Electrophoresis:

1. Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.

2. Preparation of agarose gel: To prepare 50 ml of 0.8% agarose gel, add 0.4 g agarose to 50 ml 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plate or burner by swirling the glass

beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60oC. Add 0.5 μl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

NOTE: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.

• Loading of the DNA samples: To prepare sample for electrophoresis, add 2 μl of 6X gel loading buffer to 10 μl of DNA sample. Mix well by pipetting and load the sample into the well. Load the Control DNA after extracting the DNA sample.

• Electrophoresis: Connect power cord to the electrophoretic power supply according to the conventions: Red- Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

* Molecular biology grade water is recommended.

Observation & Result

Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator.

Questions:

1. How one can remove polyphenols while isolating plant genomic DNA?

2. Mention each chemicals role for exaction of plant DNA?

3. Difference between Tris buffer, TE buffer and Tris HCl.

4. Is there any other method to isolate plant DNA? if yes mention the chemical and its role.