This document discusses nucleic acid extraction and purification methods. It outlines the organic phenol-chloroform-isoamyl alcohol extraction method. This method involves lysing the sample with Tris, EDTA, SDS, and proteinase K. Phenol-chloroform-isoamyl alcohol is added to separate the DNA into aqueous and organic layers. The DNA is then precipitated with sodium acetate and ethanol or isopropanol before being stored. The document also mentions inorganic spin column extraction and assessing DNA yield and purity.

1. NUCLEIC ACID EXTRACTION
AND PURIFICATION

2. 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

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

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

5. 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

6. Aqueous layer
Organic layer
Interface proteins

7. 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

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

9. 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

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