Solid phase DNA extraction

1. Lysis - cells are lysed using a chaotropic agent or detergent to release nucleic acid
2. Binding - nucleic acids are bound to the silica membrane
3. Washing – unwanted molecules are filtered out while nucleic acids remain bound to the
silica membrane.
4. Elution – nucleic acids are released from the silica membrane and filtered out.

Ways to break the bacterial cells open, commonly referred to as cell disruption or cell lysis to
expose the DNA within:
• Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in
the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can also
break bacterial cell walls
• Boiling in dilute sucrose, Triton X-100 detergent, Tris buffer, and EDTA after lysozyme
treatment releases DNA that can be immediately precipitated with alcohol

PK: Protease K or proteinase K or endopeptidase K is a broad-spectrum serine protease.

• It is commonly used in molecular biology to digest protein and remove contamination
from preparations of nucleic acid.
• It is stable over a wide pH range of 4-12
• Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that
might otherwise degrade the DNA and RNA during purification.

Lysis B: Salt and Tris-HCl buffer

• Chaotropic salt (e.g. guanidine HCl or guanidine thiocyanate) is added to lyse cells and
solubilize their components
• Tris or tris(hydroxymethyl) aminomethane is common biological buffer used throughout
the DNA extraction process. During cell lysis, removal of unwanted cellular components
and precipitation, tris is used to maintain a stable pH. Additionally, it plays a particularly
important role in cell lysis.
• Tris HCl is the acidic buffer. It reduces the chance of overshooting the pH. It prevents the
need to use a strong acid to adjust pH. It helps maintain reproducibility.

Incubation (or protease, beta-mercaptoethanol)

• Used for samples with high cellular and protein content to help break open cells and to
denature and degrade proteins, including nucleases

Ethanol (or isopropanol) is the binding buffer

• Added to optimize the pH and salt conditions for binding nucleic acids to the silica
membrane of the spin column.
Salt (guanidine HCl or guanidine thiocyanate)
• Facilitates the binding of nucleic acids to the silica membrane

Centrifugation
Forces solutions to go through and interact with the silica membranes

Wash buffer 1 (high-salt solution)

Wash buffer (low-salt solution)
• contains ethanol
• added to remove contaminants

Elution buffer (nuclease free water)

• with low salt concentration or nuclease free water
• Added to unbind nucleic acids from the silica membrane