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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Isolation of deoxyribonucleic acid (DNA)
 
Nucleic acids (DNA and RNA) are vital macro-molecules in all living cells. The DNA contains the basic genetic information. As such cellular DNA is located at the site of primary genetic activity (nucleus) within the cell. In prokaryotic cells, genetic activity occurs throughout the cell while in eukaryotic cells it lies in discrete particles within the cells. Most of the DNA of eukaryotes exists in the nuclei and the remaining DNA in the partially self-duplicating mitochondrial and chloroplast particles. The nuclear DNA combines with histone proteins in an orderly manner to form chromatin.
Extraction of DNA is done by a number of methods. The efficiency and recovery of extraction depends on the sample material, ionic conditions of the extraction medium, type of lysing agent used etc. The procedure described below is essentially that of Murmur's method.
 
 



Principle

Extraction of DNA is accomplished by the rupturing of cell wall and nuclear membrane followed by deproteinization and precipitation of the nucleic acid using ethanol.
 
 



Materials

Sample Material
Extraction Medium 0.15M NaCl, 0.1 M Na2 EDTA
 Lysozyme Solution, 10mg/mL
25% SDS Solution
5M NaClO4
Chloroform: Isoamyl Alcohol (24:1)
95% Ethanol
Saline Citrate (1x) 0.15M NaCl, 0.015M Trisodium Citrate (also 10-fold and 1/10-fold concentration)
3M Sodium Acetate
Isopropanol
 
 
Procedure
1.
Grind 2g of the sample material in 25mL of extraction medium in a pre-chilled pestle and mortar.
2.
Add 1 mL of lysozyme solution to the above suspension and incubate at 37°C for 30 min, shaking occasionally.
3.
After the incubation, complete the lysis by adding 2mL of SDS solution, heating this preparation for 10 min in a 60°C water bath, and finally cooling the solution to room temp in a bath of tap water.
4.
Add sufficient 5M perchlorate solution to the lysed preparation to a final concentration of 1M.
5.
Add an equal volume of chloroform-isoamyl alcohol (24:1) to the lysed preparation suspended in 1M perchlorate and slowly shake (30-60 oscillations/min) in a tightly stoppered flask for 30 min at room temp.
6.
Separate the resulting emulsion by centrifuging for 5 min at 10,000g at room temperature.
7.
After centrifugation, carefully pipette off the top clear aqueous phase from the coagulated protein emulsion at the interface.
8.
Place the aqueous phase containing the nucleic acids in a beaker.
9.
Gently stir the nucleic acid solution with a sterilized glass rod while slowly adding two volumes of 95% ethanol down the side of the beaker so that ethanol is layered over the viscous aqueous phase. Continue to gently stir the preparation to mix ethanol throughout the entire aqueous phase and spool all of the gelatinous, thread-like DNA rich precipitate on the glass rod.
10.
Drain off excess fluid from the spooled crude DNA by pressing the rod against the wall of beaker until no further fluid can be squeezed from the spooled preparation.
(If the squeezing is not done sufficiently, the alcohol adhering to DNA will make it difficult to dissolve DNA.)
11.
Dissolve the crude DNA on stirring the glass rod with its spool of material in 9mL of dilute (1/10 fold) saline citrate in a test tube or small beaker. If any solubility difficulty is encountered, continue working the sample to obtain an even suspension.
12.
To the even suspension, add 1mL of 3M sodium acetate, 1mM EDTA, pH 7.0 solution, transfer the preparation to a 100mL beaker, and gently swirl the sample while dripping in 5.4mL of isopropanol.
13.
If fibrous DNA is readily apparent, collect the DNA threads by stirring and spooling with a sterilized glass rod as before. If a gel-like preparation develops, add 0.5mL more of isopropanol and stir to spool the DNA threads as before.   Finally, remove excess fluid from the spooled DNA by pressing the sample against the walls of the beaker.
14.
Wash the sample in test tubes containing, in turn, 10mL of 70% ethanol and then 10mL of 95% ethanol.  Store the DNA in a stoppered tube (2°C refrigerator) as a spool submerged on the rod in 95% ethanol.
15.
Remove alcohol from the spooled DNA by blotting with a clean piece of filter paper and then dissolve the DNA by stirring the glass rod in a test tube containing 9mL of dilute (1/10 fold) saline citrate. When the DNA is dissolved add 1mL of concentrated (10x) saline citrate solution to achieve approximately a standard (1x) saline citrate concentration. This solution can be stored at 2°C with a few drops of CHCl3.
 
 
Notes
1. All glassware and solutions should be sterile.
2. Wear gloves to avoid nuclease contamination as well as to avoid contact with phenol (harmful).
 
 
References
1. Murmur, J (1961) J Mol Biol 3 208.
2. Sadasivam, S and Pandian, S (1978) Curr Sci 47 863.
 
     
 
 
     




     
 
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