A4 Extraction and Purification Flashcards
The complete set of instructions that makes up an organism is embedded in its genetic
blueprint, the ____-
DNA
The principles of DNA extraction, regardless of technology, can be summarized into three
steps:
1) opening the cell
2) separating the DNA from other biomolecules
3) purifying the isolated DNA from contaminants
Key Principles of DNA Extraction:
- Cell lysis
- Protein removal
- Precipitation
- Washing
- Resuspension
Why is DNA soluble?
because its sugar-phosphate backbone is hydrophilic
Why does DNA aggregate?
occurs due to a combination of factors, including unfavorable solvent conditions (like high salt concentration or alcohol), the presence of multivalent cations (like Mg2+), and the inherent properties of DNA itself.
an alkaline lysis buffer containing NaOH and SDS, which disrupts cell membranes, denatures proteins and DNA (both plasmid and chromosomal), and facilitates the release of plasmid DNA.
P2 Buffer
binding buffer (chaotropic salt solution)
facilitates DNA binding to silica by disrupting the hydration shell around the DNA, allowing it to bind more readily.
used to remove contaminants like proteins, salts, and other cellular debris, ensuring the purity of the extracted DNA.
Wash buffer
used to release the purified DNA from a silica membrane or column, allowing it to be collected in a soluble, usable state.
Elution buffer
Yeast: Requires zymolyase or lyticase to degrade the tough yeast cell wall before lysis.
Plants: Requires cellulase or pectinase to break down cell walls, along with mechanical disruption (e.g., bead beating or grinding in liquid nitrogen).
Ethanol or isopropanol washes: Remove salts and proteins.
Purpose of isopropanol or ethanol reagent:
Precipitates DNA by reducing solubility in aqueous solutions.
Removes excess salts and impurities.
Ensures DNA purity before resuspension in buffer or water.
a lysis buffer used to break open cells and release DNA
TL buffer
an enzyme that breaks down proteins in DNA extraction
Proteinase K Solution
It plays a crucial role in the binding process by ensuring that DNA efficiently adheres to the silica membrane in the spin column.
BL buffer
is used to reduce DNA solubility in water, aiding in DNA precipitation and purification
HBC buffer
used as a wash buffer to prevent cell lysis or shriveling, maintain osmotic balance, and as a resuspension medium for cells or enzymes.
PBS
a proprietary reagent from Omega Bio-tek, effectively removes PCR inhibitors, including humic acid, ensuring high-quality DNA suitable for downstream applications like PCR, sequencing, and next-generation sequencing.
cHTR Reagent
a buffer containing Sodium Dodecyl Sulfate (SDS), a detergent that disrupts cell membranes and denatures proteins, facilitating DNA release and isolation.
DS buffer
used to release the purified DNA from a silica column or other binding matrix, allowing for its collection in a soluble form.
Elution buffer
a lysis buffer that helps break open cells and release DNA by promoting cell lysis and protein denaturation.
SLX-Mlus Buffer
often used in soil DNA extraction kits, helps in denaturing cells, eliminating PCR inhibitors, and enhancing DNA binding to a silica membrane for purification.
XP1 buffer
is formulated to isolate high purity cellular DNA
from soil samples typically containing humic acid and other inhibitors of PCR. This
kit uses a novel and proprietary method to isolate genomic DNA from a variety of
environmental samples without organic extractions.
E.Z.N.A.® Soil DNA Kit
Buffer that precipitates inhibitors
P2 buffer
used to precipitate DNA out of solution by reducing its solubility, causing it to clump and become visible, and also helps remove salts.
ethanol
a cationic detergent, to disrupt cell membranes and complex with DNA, which then precipitates out of solution when salt concentration is adjusted.
CTAB
Genomic DNA Extraction from Plant Tissue
:
Two methods:
CsCl Gradient Purification: More labor-intensive but yields highly purified DNA.
CTAB-Based Extraction: Simpler, scalable, and higher yield, but less pure.
High-salt precipitation method for faster but smaller DNA fragments.
Key reagents:
CTAB: Disrupts membranes and precipitates polysaccharides.
NaCl: Removes protein contaminants and stabilizes DNA.
Tris buffer: Maintains pH stability during extraction.
EDTA: Chelates divalent cations (Mg²⁺, Ca²⁺) to inhibit DNase activity.
β-Mercaptoethanol: Reduces oxidation of polyphenols.
PVP: Binds and removes polyphenols.
Phenol-Chloroform-Isoamyl Alcohol: Removes proteins and polysaccharides.
Isopropanol/Ethanol: Precipitates DNA.
Final storage: DNA is typically stored in TE buffer to maintain stability.
Silica spin columns are used in DNA extraction kits because the silica material selectively binds to DNA under specific conditions, allowing for efficient purification by separating DNA from other cellular components like proteins and lipids.
