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DNA Extraction, Eng Lang

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15 views

DNA Extraction, Eng Lang

All details given

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akashpal.dms.1211
Copyright
© © All Rights Reserved
Available Formats
Download as PDF or read online on Scribd
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yy ; f CHAPTER Isolation of DNA from Plant Material EXPERIMENT 5 Objective ip of papayal To isolate DNA from available plant matetial (such as spinach leaves, green pes see PUP oe banana, etc.) Materials required ated beakers of 200 mL each; one Beakers: A lange beaker of 500 mL capacity or flask of 250 mL, 2 gradu a + cafe stand: beaker of 100 mL; one graduated cylinder of 100 mL; fermometer; clock or wate test tubes, tes on d 95% ethyl dropper; blender or mortar; table salt 2 8 refrigerator liquid soap; 91% isopropyl ee i aap eresh spinach leaves/pea seeds/pulp of papaya or Banand! strainer or che lass rod /chopstick/toothpick (ong); cold water and hot water. Procedure DNA extraction process involves the following steps: Cell disruption or cell lysis @ Removing membrane lipids Removing proteins DNA precipitation i f (Note: Tis technique is basically responsible. for the extraction of both types dpe ie DNA ae a Since, no enzyme is sed forthe digestion or remocd! of RNA, the white threa aaeited RNA. Therefore, pure DNA isnot isolated by this technique.) 1. Cell disruption or cell lysis Take 50 g or half cup of finely chopped © Place the spinach in the blender, add a t toit. Blend on high speed for 15 minutes. @ Filter the mixture through a strainer or che Fill the beaker about to half. 2. Removal of membrane lipids ‘Add 2 tablespoons (10 mL) of liquid detergent to the filtered mixture, & Stir the mixture gently for proper mixing. Allow the mixture to stay undisturbed for 10 minutes. spinach. Wash it with hot water. easpoonfull of table salt and 200 mL or 1/3rd cup of cold water ese cloth into a large beaker or flask. 3. Removal of proteins © Pour 5 mL of the mixture in a small beaker and add a pinch of enzyme protease or one-fourth teaspoon of meat tenderizer. The enzyme removes proteins from the solution, which settle down at the bottom. © Stir the mixture gently. Do not stir too hard, otherwise, it will break the DNA into small pieces. -_ 4, DNA Precipitation ¢ Tilt the test tube and slowly pour 10 ml or mote of ice-cold ethanol of isopropanol down the side of it go that it forms a layer on the top of spinach mixture f Place the test tube in the refrigerator for 5 minutes. E @ Remove and wait for another 5 minutes i ¢@ DNA rises to the top of the test tube and a i i i sa. Spool the DNA using a tooth pick or chor a as mucus-like white strands in the alcohol layer. 4+ Millions of DNA strands get clumped on the tip of toothpick or chopstick ¢@ DNA can be collected with a wire hook or dropper and placed on paper towel. Put the DNA into a small container having about 2-3 mL of TE buffer and store it in the refrigerator. Addition of ethanol to the mixture for precipitation of DNA ‘pooling of DNA Fig. 5.1 Process of DNA extraction Precautions 4 Use right amount of spinach and right amount of meat tenderizer. Wrong proportion can-tffées the results. Do not stir the solution too hard. This breaks the DNA and DNA fails to form the mesh in alcohol. @ Add twice as much cold water as the DNA source. Alcohol should form a layer on top of the spinach extract. Alcohol being less dense than water forms a separate layer at the top. (Note: Other soft plant material like broccoli or onion can also be used to get plenty of DNA.)

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