wk 3 Flashcards
(29 cards)
first step in laboratory
procedures required to perform any chosen molecular applications.
NUCLEIC ACID EXTRACTION
Key Steps:
Nucleic Acid Extraction
Key Steps:
Sample preparation 1.
Cell lysis 2.
Nucleic acid purification 3.
Quantification and quality assessmen
Initial release of the cellular material by breaking the cell wall (if present), cell and nuclear membranes.
cell lysis
cell lysics
Mechanical: Bead beating, sonication.
Chemical: Detergents (SDS), alkaline lysis.
Enzymatic: Proteinase K, lysozyme
Purification
Organic extraction (phenol-chloroform): Effective but toxic.
Silica-based columns: Fast and highly pure.
Magnetic bead separation: Automation-friendly
DNA purity check
Spectrophotometry
Key Components in Organic Isolation
Key Components in Organic Isolation
Phenol and Chloroform: Dissolve hydrophobic components.
High Salt & Low pH: Help in phase separation.
RNase: Eliminates RNA contamination.
Cetyltrimethylammonium bromide (CTAB): Removes polysaccharides from
fungal samples
Organic Isolation Methods
1 Cell lysis
- Addition of organic solvent
3 Centrifugation
4 DNA precipitation &Recovery
5 Pellet washing 3 Rehydration
Denatures proteins and dissolves hydrophobic impurities
Phenol
Helps separate aqueous and organic phases.
Chloroform
Facilitates separation of contaminants from DNA
Biphasic Emulsion:
Addition of Organic Solvent:
Phenol-chloroform to create biphasic emulsion
Phenol: Denatures proteins and dissolves hydrophobic impurities.
Chloroform: Helps separate aqueous and organic phases.
Biphasic Emulsion: Facilitates separation of contaminants from DNA
Centrifugation phases
Hydrophilic phase (top)/ Aqueous: DNA.
Hydrophobic phase (bottom): Lipids, proteins.
Interface: White precipitate of amphiphilic molecules.organic solvents
how to precipitate and recover dna and what ratio should theybe
Using ethanol or isopropanol and salts.
2:1 ethanol or 1:1 isopropanol ratios facilitate DNA precipitation
Salt Additives:
`Ammonium, potassium, sodium acetate/chloride
Enhance recovery of low-DNA samples.
Carrier Molecules:
Carrier Molecules:
Glycogen, yeast RNA, linear polyacrylamide.
:
Derived from mussels, potential DNA contamination.
Commercial versions treated with DNase to remove unwanted nucleic acids
Glycogen
Optimal Carrier Amount for glycogen
Optimal Carrier Amount: 10–20 µg per mL of isopropanol mixture.
- Pellet Washing & Rehydration:
70% ethanol wash, then resuspension in TE buffer or water.
Centrifugation of precipitated DNA.
Pellet Collection:
Salt Removal:
Rinse with 70% ethanolSalt Removal:
- protects the DNA from damage by DNases for long-term storag
EDTA
Rehydration Buffers:
- TE buffer (10 mM Tris, 1 mM EDTA).
- EDTA - Nuclease-free water
