NUCLEIC ACID EXTRACTION AND PURIFICATION 2023.pptx

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NUCLEIC ACID EXTRACTION
AND PURIFICATION

LESSON LEARNING OUTCOMES
• Identify the types of specimen from which DNA and RNA can be
isolated for further analysis
• Explain the steps involved in NA extraction
• Differentiate between the different methods of NA extraction

Purpose of NA Isolation?
Good yield  [ ] of DNA
Good quality/pure  free from
contamination

DNA EXTRACTION METHODS
ORGANIC
Phenol-Chloroform-Isoamyl-
Alcohol Method
INORGANIC
SPIN COLOUM METHOD

Phenol-Chloroform-Isoamyl-Alcohol Method
Sample: Blood, tissue, bacteria/fungal culture
Add lysis buffer: Tris, EDTA, SDS, pH 8.0, Proteinase K
Pellet  Add phenol-chloroform-isoamyl alcohol
(25:24:1) repeat step
Add Chloroform-isoamyl alcohol (24:1)
Spin and mix
Spin and mix
Spin and mix

Aqueous layer
Organic layer
Interface proteins

Phenol-Chloroform-Isoamyl-Alcohol Method
Continues… (DNA precipitation)
Add Chloroform-isoamyl alcohol
Sodium Acetate
Add Ice Cold Isopropanol/Ethanol
Store DNA in Tris EDTA buffer
Store @ 4°C, -20°C or -80°C
Spin and mix
Spin and mix

Lesson Summary
https://www.aatbio.com/resources/assaywise/2022-11-1/overview-of-dna-extraction-methods

END OF LESSON QUIZ
What is the role of the following chemicals in NA extraction?
• EDTA
• Tris
• SDS
• Phenol
• Chlorophorm
• Proteinase K
• Sodium acetate
• Isopropanol

Next Lesson….
• Inorganic extraction method (spin column)
• Quantitative and Qualitative Analysis of DNA Yield and Purity

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