MOL BIO LAB L6 (Midterms- DNA & RNA Extraction) Flashcards
One of the most pivotal steps in molecular biology, being routinely used in many areas of the biological and medical sciences, as this procedure marks a starting point in any molecular diagnostic kit.
Nucleic acid extration
Encompass extraction of both DNA and RNA but can be more broadly characterized into chemically driven or solid phase methods.
Nucleic Acid Extraction (NAE) methods
These methods rely on biochemical properties of the cellular components to elicit the desired molecular separation and might exhibit preference or exclusivity in extracting DNA or RNA, depending on its intrinsic characteristics.
Chemical methods of NAE
Enumerate the four techniques under the chemical method of NAE
- Osmotic shock
- Enzymatic digestion
- Detergents
- Alkali treatment
The only chemical technique that the mode of lysis is HARSH
Alkali treatment
Principle of Osmotic Shock
Osmotic rupture of membrane
The cost is moderate and is usually applied for general use
Detergents
Principle of Enzymatic digestion
Digestion of cell wall
Principle of Detergents and Alkali treatment
Solubilization of membrane
Cheap at small scale; expensive at large scale
Enzymatic digestion
Used for spheroplasts and protoplasts
Osmotic shock
Used for gram-positive and gram-negative bacteria
Enzymatic digestion
The cost of these chemical techniques is CHEAP
Osmotic shock and alkali treatment
Used to extract DNA from bacteria and is based on the phenomenon of buoyant and specific density
Cesium Chloride (CsCl) Gradient Centrifugation with Ethidium Bromide (EtBr)
Principle of Ultrasonication or cavitation
Disruption of cells by pressure
Principle of Pressure cell (“French press”)
Disruption of cells by shear force
The separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of GUSCN, sodium acetate, phenol, and chloroform, followed by centrifugation.
Guanidinium Thiocyanate-Phenol-Chloroform Extraction
Enumerate the techniques under the Mechanical methods of NAE
- Homogenization (blade or pestle)
- Ultrasonication or cavitation
- Pressure cell (“French press”)
- Ball mill
Principle of Homogenization (blade or pestle)
Shredding of cells
Principle of Ball mill
Cells crushed between glass/steel/balls/beads
The only mechanical technique that has a MODERATE mode of lysis
Homogenization (blade or pestle)
The only mechanical technique that costs CHEAP
Ball mill
A mechanical technique that costs moderate (method of choice for large scale)
Homogenization (blade or pestle)
A mechanical technique that costs moderate to expensive
Ultrasonication or cavitation
A mechanical technique that is used for animal tissues
Homogenization (blade or pestle)
To evaluate DNA purity, measure absorbance from_______ to _______ to detect other possible contaminants.
230nm; 320nm
A mechanical technique that is used for Gram- negative and some and Gram positive bacteria
Pressure cell (“French press”)
A mechanical technique that is good for spheroplasts but not primary cells
Ultrasonication or cavitation
A mechanical technique that used for bacteria, yeast, microalgae, unicellular animal cells
Ball mill
The most common purity calculation is the ratio of the absorbance at?
260nm divided by the reading at 280nm. (260nm/280nm)
The most common technique to determine DNA yield and purity is measurement of?
Absorbance
What are needed for the absorbance method?
- Spectrophotometer equipped with a UV lamp
- UV-transparent cuvettes (depending on the instrument)
- Solution of purified DNA
Where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution.
260nm (A260)
Absorbance readings are performed at?
260nm (A260)
Good-quality DNA will have an A260/A280 ratio of?
1.7-2.0
A reading of ____ does not render the DNA unsuitable for any application, but Iower ratios indicate more contaminants are present.
1.6
DNA purity formula
A260/A280
Formula used for correcting for turbidity
DNA purity (A260/A280)= (A260 reading — A320 reading) / (A280 reading — A320 reading)
Can indicate that organic compounds or chaotropic salts are present in the purified DNA.
Strong absorbance around 230nm
This ratio can help evaluate the level of salt carryover in the purified DNA.
ratio of 260nm to 230nm
In terms of absorbance, the _____the ratio, the _____ the amount of thiocyanate salt is present.
lower; greater
The A260/A230 is best if greater than?
1.5
A reading at ____ will indicate if there is turbidity in the solution, another indication of possible contamination.
320nm
One optical density unit (or absorbance unit) at 260 nm is equivalent to?
50 mg/L (or 50ug/mL) of double-stranded DNA and 40 ug/mL of RNA.
Formula for DNA Concentration (ug/mL)
(A260 reading — A320 reading) x dilution factor x 50ug/ml
NOTE: DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50ug/ml pure dsDNA.
Formula for RNA Concentration (ug/mL)
(A260 reading — A320 reading) x dilution factor x 40ug/ml
DNA yield (ug) formula
DNA concentration x total sample volume (ml)
This is the limit for suitability in terms of purity
2.5
In Guanidinium Thiocyanate-Phenol-Chloroform extraction, this can be recovered through precipitation by isopropanol and can be used for subsequent process.
Total RNA
In Guanidinium Thiocyanate-Phenol-Chloroform Extraction, ______ remains in the upper aqueous phase, while most of ______ and proteins part remain either in the interphase or in the lower organic phase under acidic condition.
Total RNA; DNA
Dedicated to plasmid DNA isolation
Alkaline Extraction
Used in the field of forensics for DNA extraction from various sources, such as hair, blood stain cards, and buccal swabs
Chelex® Extraction
Inhibits DNA degradation by chelating metal ions which cause DNA breakdown at high temperature and lower ionic conditions
Chelex® Extraction
What is the basic principle of Alkaline Extraction?
selective alkaline denaturation of high molecular weight chromosomal DNA, while covalently bond circular plasmid DNA remains intact.
Alkaline Extraction involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of ______and ______ ).
SDS or sodium dodecyl sulfate; NaOH (sodium hyroxide)
Poly(A) RNA hybridizes with an oligo(dT)-cellulose matrix, under high-salt conditions
Purification of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography
Based on liquid and stationary phases, which selectively separate the target analyte from the solution based on specific hydrophobic, polar, and/or ionic properties of both solute and sorbent.
Solid-Phase Nucleic Acid Extraction
Types(?) of Solid-Phase Nucleic Acid Extraction
- normal/regular SPE
- reverse SPE
- ion exchange SPE
Enumerate the solid phases of Solid-Phase Nucleic Acid Extraction use:
- silica matrices
- Glass Particles
- Diatomaceous Earth
- Magnetic Beads-Based Nucleic Acid Purification (most common)
- Anion Exchange Material
- Cellulose Matrix
