This document discusses the key steps in isolating genomic DNA from cells: 1) Cell lysis using a buffer with SDS to break open cell and nuclear membranes and release DNA. 2) Addition of protease to degrade proteins surrounding the DNA. 3) Precipitation of DNA by adding salt to neutralize charges on DNA molecules followed by cold alcohol, which causes the DNA to clump out of solution.

1. Relevance of DNA isolation
• Isolation of DNA is often the first step before further
analysis
• DNA profiling (forensics)
• cloning
DNA isolation • disease diagnosis
• DNA sequencing
• genetically modified organisms (GMO) - agriculture,
pharmaceutical
• Environmental testing, bioterrorism
Structure of the cell Extraction of genomic DNA
• Plasma membrane and membranes of organelles nuclear • Cell collection
envelope included • Add Lysis buffer to cells to break open cell and nuclear
• DNA located in nucleus membranes and release nuclear contents
• A lot of proteins around • Digest sample with protease to degrade proteins
• Mitochondrial DNA • Precipitate DNA with cold alcohol in high salt
Lysis buffer Why add protease?
• Lysis buffer CH3
• Protease destroys nuclear proteins that bind DNA and
• 50 mM Tris-HCI, pH 8.0 to maintain the pH CH2 cytoplasmic enzymes that breakdown and destroy DNA
of the solution at a level where DNA is stable CH2
• Protease treatment increases the amount of intact DNA
• 1% SDS to break open the cell and nuclear
CH2
CH2 that is extracted
membranes, allowing the DNA to be released CH2
into the solution (SDS also
CH2
CH2
denatures and unfolds proteins, CH2
making them more susceptible CH2
CH2
to protease cleavage) CH2
O
O O
S
O-
SDS
1

2. Adding salt Precipitation of DNA
• The addition of NaCI allows the • DNA does not dissolve in alcohol.
O
DNA molecules to come together Na O P O • Addition of cold alcohol makes the DNA clump together
instead of repelling each other, +
Na+ O
Base
and precipitate out of solution
thus making it easier for DNA to CH2 O • Precipitated DNA molecules appear as long pieces of fluffy,
precipitate out of solution when Sugar stringy, web-like strands.
alcohol is added
• Microscopic oxygen bubbles “aggregate”
• Na+ ions bind to the phosphate O
groups of DNA molecules,
Na+ together, as the DNA precipitates
O P O Base
neutralizing the electric charge of
Na+
O • The larger, visible air bubbles “lift” the
the DNA molecules CH2 O DNA out of solution, from the aqueous
Sugar
into the organic phase
• Our protease solution already • The DNA in the glass vial can last for
contains salt OH
years
2