dna extraction from whole organism

8/3/2019 DNA Extraction From Whole Organism

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-Contains the genetic instructions used inthe development and functioning of livingthings

-Its is different from RNA in two ways; RNAcontains ribose instead of a deoxyribose, and

uracil in place of thymine residue in DNA.


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-In tissues with high nuclear to cytoplasmicratio

-Example: Thymus, liver, spleen, muscletissues.


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-Soluble in weak alkali solution

-Insoluble in alcohol


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Phosphodiester bond joins nucleotides

Hydrogen bond between base pairs of oppositechains

Van der Waals between stacked bases


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1. Disruption of Cell Membrane

- to release nucleic acid inside the cell

2. Nuclease Inactivation- protein denaturation


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3. Dissociation of Nucleoproteins in DNA

-use of solvents, detergents, phenols andenzymes

4. Precipitation of Nucleic Acid

-through ethanol


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1. Liquid Nitrogen

-freezing sample quickly

-preserve DNA and disable digestive enzymes

2. Homogenization

-disrupt cell membrane

-circular motion to avoid mechanical stress


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3. Tris-HCl ph 8.0

-DNA soluble at slightly alkaline solution

-pH stabilizes interactions of DNA

-reduces ionic interaction of positivelycharged histones and negatively charged

backbone of DNA.


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4. Meat tenderizer-protease (e.g. bromelain) optimal pH is 4.5-5.5

-break down proteins by hydrolysis of peptidebonds


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5. 10% SDS

-denaturation of proteins and enzymedeactivator

-binds with + charged histones that reduceaffinity with DNA strands

-disruption of ionic interaction of histonesand DNA.


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6. Heat at 55C for 1 hour

-denaturation of proteins-must not be too hot, at 80-90C,

DNA starts to unwind

7. Organic solvent (THF)

-Lipids, proteins soluble in organic

-Nucleic acid soluble in aqueous


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8. 0.5M NaCl

-for complete dissociation of DNA-histone-decreases solubility of DNA

9. Cold 95% ethanol

-DNA is highly insoluble inorganic solvents

-ethanol lowers dielectric constant

of water-low temp decreases solubility of

enzymes


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10. Tris-EDTA buffer ph 8.0

-prevents denaturation

-inactivates enzymes

-inhibit growth of microorganisms

-forms compelexes with the metal components ofthe secondary structure of the DNA such as Mg2+


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Results:

Wet mass: 0.089g

Absorbance at 260nm: 0.162

Absorbance at 280nm: 0.112

Ratio: 1.45

% purity: 99.25%

DNA concentration: 72.5 g/mL


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A280/A260 %Nucleic Acid A280/A260 %Nucleic Acid

1.75 0.00 0.87 5.0

1.63 0.25 0.85 5.5

1.52 0.50 0.82 6.0

1.4 0.75 0.80 6.5

1.36 1.00 0.78 7.0

1.3 1.25 0.77 7.5

1.25 1.50 0.75 8.0

1.16 2.00 0.73 9.0

1.09 2.50 0.71 10.01.03 3.00 0.67 12.0

0.98 3.50 0.64 14.0

0.94 4.00 0.62 17.0

0.60 20.0


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dna extraction from whole organism

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