DNA EXTRACTION Flashcards by Kyra Catedral

DNA EXTRACTION

• Goal:

To obtain pure and good quality DNA

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• separates DNA from cellular fluid & cellular debris such as proteins, lipids, polysaccharides & other compounds.

DNA EXTRACTION

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• the technique used to isolate DNA in a biological sample.

DNA EXTRACTION

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Genetic analysis:

Scientific research
Medical diagnostics
Forensic

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■ Gene cloning – introduction of DNA into cells (introduction of DNA into cells (humans, animals or plants) for diagnostic purposes

Scientific- Research

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Medical- Diagnostics

■ Genome sequencing (outbreak analysis)
■ Hereditary & infection disease
■ Vaccine development

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Forensic-

Paternity, etc.

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DNA Isolation:

aims to collect DNA by physical or
chemical means.

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DNA Purification:

the process of eliminating
contamination from isolated DNA.

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DNA Extraction:

achieve both purification and isolation.

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Where do we obtain DNA from? From all living things such as:

● Human cell
● Plant cell
● Bacterial cell

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Basic Steps

Cell lysis
Precipitation
Purification

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breaking cells to release DNA from nucleus

Cell Lysis

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Separates DNA to cellular debris
a. Such as proteins, RNAs, other macromolecules

Precipitation

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to remove remaining unwanted material

Purification

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General Steps for DNA Extraction (5)

1- Tissue Homogenization / Cell Disruption / Cell Lysis

2- Denaturation and Separation of Other Biomolecules from the Nucleic Acid

3- Precipitation of Nucleic Acids

4- Washing of Precipitated Nucleic Acids

5- Drying of Pellet and Dissolution of Dried Pellet

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TISSUE HOMOGENIZATION OR CELL DISRUPTION OR LYSIS

• Mechanical Methods
• Chemical Treatment
• Enzymatic Treatment
• Thermal

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MECHANICAL METHODS (MVSHF)

• Mortar and pestle
• Vortexing with beads
• Sonication
• High pressure
• French press

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CHEMICAL TREATMENT

• Denaturants
• Reducing agents
• Buffer
• Cell Lysis agents
• Salt

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• ENZYMATIC TREATMENT (PCLL)

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• Protienase K: animal cells
• Cellulase: Plant cells
• Lyticase: Yeast
• Lysozyme: Bacteria

(With the use of Temperature)

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THERMAL

Factors that Influence the Disruption Strategy (SSPCC)

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• Stability of Molecules
• Size of the Sample
• Presence of Inhibitors
• Cohesion of Cells
• Cell Membrane Type

Denaturation and Separation of Other Biomolecules from the Nucleic Acid (CEC)

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Chemical Treatment
Enzymatic Treatment
Centrifugation

Denaturation and Separation of Other Biomolecules from the Nucleic Acid

Chemical Treatment (SPP)

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○ Sodium dodecyl sulfate
○ Phenol and chloroform
○ Protein denaturation

Denaturation and Separation of Other Biomolecules from the Nucleic Acid (CEC) ● Enzymatic Treatment

○ Protease; Proteinase

Precipitation of Nucleic Acids MONOVALENT CATIONS

Ammonium, Potassium or Sodium Acetate or Lithium or Sodium Chloride Alcohol Precipitates Nucleic Acid ETOH (95% Absolute) Isopropanol

________, like sodium ions, neutralize the negatively charged phosphate groups on nucleic acids, overcoming electrostatic repulsion, enabling the aggregation of nucleic acid molecules, and ultimately promoting their precipitation for subsequent purification.

Monovalent cations

Precipitation of Nucleic Acid Alcohol precipitates nucleic acid

○ ETOH (95% - Absolute) ○ Isopropanol

Washing of Precipitated Nucleic Acids (2)

● 70-80% ETOH ● Centrifugation

Drying of Pellet and Dissolution of Dried Pellet Drying: (2) ● Dissolution in_____ or ____ ● Room temperature or at____

● Air drying or vacuum crying ● sterilized molecular grade water or TE ● 50-55 C water bath

• Resuspend in TE • Neutral pH • Chelating Agent

Nucleic Acid Stabilization

Nucleic Acid Stabilization • Store at______ • Aliquot sample • AVOID:______ • Use_____ free reagents and plastic ware

- 20 C or - 70 C freeze-thaw cycles nuclease

PHENOL CHLOROFORM EXTRACTION • The nucleic acid solution is extracted by successively washing with a volume of_______; a volume of______:______:______, and_____:_____ • Centrifugation is performed intermittently and the upper aqueous phase is transferred to a new tube while avoiding the interphase • The contaminants are denatured and accumulate in the organic phase and the nucleic acids are preserved in the aqueous phase Anotherway of removing proteins is by using the enzyme proteinase K

phenol phenol: chloroform: isoamyl alcohol (25:24:1) chloroform: isoamyl alcohol (24:1)

PRECIPITATION OF NUCLEIC ACIDS •____ precipitation • Diluting nucleic acid with a_____, adding alcohol to it and mixing gently • The precipitated DNA is pelleted by____ • The salts and alcohol remnants are removed by washing with______ • _____ and______are the standard alcohols used for Nucleic Acid extraction

Alcohol monovalent salt centrifugation 70% alcohol Ethanol and Isopropanol

RESUSPENDING DNA • Nucleic Acid pellet resuspended in either_____ or_____

sterile distilled water or Tris-EDTA (10mM Tris: 1mM EDTA)

PURIFICATION OF DNA • DNA is purified by incubating the Nucleic Acid solution with _______ at_____ and reprecipitation following________ extraction to remove the RNAse

RNASe A (10mg/mL) at 37C phenol: chloroform

SPECIFIC PROCEDURES

1. Disrupt the Cell 2. Disrupt Protein Disulphide Bonds 3. Denature Proteins Further, Remove Lipids and some Polysaccharides 4. Precipitate Nucleic Acids 5. Washing Nucleic Acid 6. Remove RNA and Elute 7. Resuspend DNA 8. DNA Measurement

1. Disrupt the Cell • Preheat CTAB isolation buffer to_____ in a water bath • Weigh 0.1g fresh, dried, or frozen material • Place in a______ • Add _____and grind to fine powder. • Optional: Add______ and grind further • Transfer powdered sample to 2mL tube microfuge tube

65C mortar and pestle liquid nitrogen 40mg PVPP(Polyvinylpolypyrrolidon)

2. Disrupt Protein Disulphide Bonds • Add 800uL CTAB isolation buffer • Vortex to mix • Incubate at 65C for 1 hour with occasional swirling or shaking

3. Denature Proteins Further, Remove Lipids and some Polysaccharides • Add 800 uL______ • Mix gently for 10 minutes at room temperature • Centrifuge at room temperature (6000 g for 10 minutes) • Pipet the upper aqueous phase with wide bore pipet • Place in a clean 2mL microfuge tube

chloroform-isoamyl alcohol (24:1)

4. Precipitate Nucleic Acids • Add 600uL cold_____ • Mix gently by inverting tubes several times • Incubate several hours to overnight at room temperature • Centrifuge for 1-2 minutes at 12000 rpm • Gently pour off the supernatant

isopropanol

5. Washing Nucleic Acid • Add 800uL____ directly to pellet. Swirl gently. Leave for at least 20 minutes with gentle swirling. • Centrifuge at 12000 rpm for 3 minutes. Pour off supernatant carefully. • Air dry briefly by inverting tubes in paper towel at room temperature

ETOH

6. Remove RNA and Elute a. Add 100uL high salt TE and 1 uL RNAse. Incubate 30 minutes at 37C. b. Dilute sample with: i. 200uL 1XTE ii. 10uL 3M sodium acetate (5.2) iii. 250uL cold 70% ETOH then gently mix to precipitate DNA

7. Resuspend DNA • Resuspend in 20uL TE buffer (10mM Tris-HCl, 1mM EDTA, pH8) to dissolve precipitate • Store at -20C to -40C until further use

8. DNA Measurement • Measure DNA yield using______ at _____

UV-Visible Spectrophotometer at 260 nm

Centrifugation is performed intermittently and the upper____ phase is transferred to a new tube while avoiding the____

aqueous interphase

• The contaminants are denatured and accumulate in the_____ phase and the nucleic acids are preserved in the_____ phase

organic aqueous

Anotherway of removing proteins is by using the enzyme…

proteinase K

DNA EXTRACTION Flashcards

(48 cards)