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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Extraction of plant total DNA (Rapid method)
 
There are a variety of procedures described for the extraction of total DNA from plant tissues. These procedures, however, include removal of proteins by phenol or similar agent. The method described below excludes use of such reagents. Furthermore, it is rapid and hence a large number of samples can be extracted in a unit time. This method is more suited to rice leaves and other similar starting materials. The DNA obtained is suitable for restriction digestion analysis.
 
 



Principle

The DNA is extracted using a suitable extraction medium and relieved of carbohydrate and other bulk impurities by adding potassium acetate and the DNA is precipitated with isopropyl alcohol
 
 
Materials
Plant material
Extraction buffer
   50mM Tris-HCl (pH 8.0)
   50mM EDTA NA2+
   250mM NaCl
   15% Sucrose
Ethanol
sopropanol
5M Potassium acetate solution
RNase
Sodium acetate 3 M
TE buffer
   10mM Tris-HCl
   1mMEDTA
 
 


Procedure
1.
Wash the plant material in running tap water followed by sterile distilled water. Remove the water on the material by blotting with a filter paper and cut into small bits, if necessary.
2.
Weigh out 3-5g of the above material and transfer to a suitable clean dry porcelain pestle and mortar. Grind the material in the presence of liquid nitrogen to a fine powder. Do not allow the powder to thaw.
3.
Using a metal spatula, transfer the frozen powder to a 50mL tube/conical flask containing 15mL extraction buffer maintained at 65°C in a water bath. Mix well with the spatula and by inverting the tube. Incubate the mixture at 65°C for 15 min with intermittent gentle shaking.
4.
Add 5 ml of 5 M potassium acetate solution, mix vigorously and incubate on ice for 20 min.
5.
Centrifuge the content at 4,000 rpm for 20 min.
6.
Filter the supernatant (containing DNA) through 2 layers of fine cloth. Collect the filtrate in another tube/flask. If the filtrate is green/brown add 5 mL of 5 M potassium acetate solution and repeat steps 5 and 6.
7.
Add 2/3 volume of isopropanol to the filtrate (2 mL isopropanol to 3 mL filtrate) and shake tube slowly by tilting it. Incubate the tubes at -70°C for 30 min or -20°C overnight to precipitate DNA.
8.
Hook out DNA if possible using a Pasteur pipette with a curved tip. Otherwise, pellet DNA by centrifuging at 10,000 rpm for 15 min. Wash the pellet with ice-cold 70% ethanol followed by absolute ethanol. Dry the pellet in vacuo.
9.
Suspend the DNA pellet in TE buffer. The DNA preparation can be further purified as given below:
10.
Add 10mlof RNase (10 mg/ml) to the DNA solution and incubate at room temperature for 15-30 min to remove RNA impurity.
11.
Add 1/10 volume of 3 M sodium acetate and 2 volumes of 95% ethanol. Mix gently to precipitate the DNA.   Incubate at -20°C for 1h to increase the yield of DNA precipitation.
12.
Hook DNA precipitate, if possible, or collect by centrifugation (Step 8).
13.
Resuspend the DNA pellet in TE or suitable buffer for further use. It can be stored at 4°C for some weeks but should be frozen at -20°C for long term storage.
 
 
Notes
1. The ratio of powdered tissue to the extraction buffer is important. If the mixture is too thick and do not mix well when tube is inverted, increase the volume of buffer until a thick liquid is obtained. When more buffer is required increase the volume of 5 M potassium acetate.
 
 


References
1. Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983). A plant DNA minipreparation: version II. Plant Molecular Biology Reporter I (4): 19-21.
 
     
 
 
     




     
 
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