Plant Lab Protocols / Methodology for Nucleic Acids
Extraction of plant DNA
A number of methods are available for the extraction of plant DNA depending upon the starting tissue, homogenization conditions etc. The method below is ideal to obtain DNA from plant sources in a relatively easy way.
Materials
» Seedlings or any other fresh tissue
» Liquid nitrogen
» Extraction buffer
100mM Tris-HCl (pH 7.8) 6.06g
10mM EDTA Na2 9.30g
500mMNaCl 14.61g
Water 500mL
» 5M Potassium acetate
Dissolve 24.55g in 50mL water, sterilize and use.
» 3M sodium acetate
Dissolve 2.46g anhydrous salt in 10mL water, sterilize and use.
» Suspension buffer
50mM Tris-HCl (pH 8.0) 0.61g
10mM EDTA Na2+ 0.37
Water 100mL
» TE buffer
10mM Tris-HCl (pH 7.5) 0.12g
1mM EDTA Na2 0.37g
Water 100mL
Method
1.
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Weigh 5g of plant tissue, quickly freeze in liquid nitrogen and grind to a fine powder in a pestle and mortar.
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2.
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Add 75mL of extraction buffer in small aliquots and grind thoroughly.
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3.
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Transfer the homogenate to a 250mL flask. Add 5mL of 20% SDS and mix thoroughly using a magnetic stirrer for 15-20 min. Then incubate the contents at 65°C for 10 min.
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4.
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Add 50mL potassium acetate solution, mix and incubate at 0°C for 30 min in order to precipitate proteins and polysaccharides.
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5.
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Remove the precipitate by centrifuging at 25,000g for 15 min. To the supernatant add six tenth volume of isopropanol and stand -2C C forat least 30 min to precipitate DNA.
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6.
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Pellet DNA at 20,000g for 15 min. Discard the supernatant and drain off any liquid by inverting the tubes on filter paper for 2-3 min.
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7.
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Redissolve DNA pellet in suspension buffer (3mL). Add 1.8mL isopropanol and 18QaL 3M sodium acetate solution and let stand at -20°C for 1h.
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8.
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Repellet DNA by centrifugation, wash with ice-cold 80% ethanol andgently dry in vacuo or streaming nitrogen gas.
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9.
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Redissolve the DNA pellet in a suitable volume of (0.5-5 mL) TE buffer.
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10.
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Estimate the DNA content and check purity by UV spectrometry.
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DNA Purification by CsCl Centrifugation
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11.
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To 1mL of DNA solution (Step 9) add 0.75g cesium chloride and dissolve completely. Add 10/* L of ethidium bromide (10 mg/mL) in TE buffer. Transfer the contents to a suitable ultracentrifuge tube (for example, Beckman SW 50.1 rotor).
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12.
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Centrifuge at 1,80,000g and at 15°C for 16-24h in an ultracentrifuge.
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13.
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Collect the DNA band using a Tasteur pipette or syringe' viewing in a UV transilluminator.
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14.
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Extract out the ethidium bromide with small volumes of water-saturated n-butanol until the extract is colourless. Mix the aqueous extract with an equal volume of TE buffer.
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15.
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Precipitate DNA by adding an equal volume of isopropanol at -20°C for 1h.
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16.
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Pellet the DNA by centrifugation, wash with ice-cold 80% ethanol, dry and resuspend in a small volume of suitable or TE buffer.
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Notes
1.
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A good DNA preparation generally exhibits the following spectral characteristics:
A 230 = < 0.1; A 230/A 260 < 0.45; A 280/A 260 < 0.55. The extinction coefficient of DNA is E1cm1% = 200 or DNA at a concentration of 50mglmL shows an absorbance of 1 at 260nm.
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2.
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Store aqueous DNA preparation frozen at -70°C; for longer duration store under ethanol.
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