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SECOND SEM 3rd YEAR > MOL BIO LAB L6 (Midterms- DNA & RNA Extraction) > Flashcards

MOL BIO LAB L6 (Midterms- DNA & RNA Extraction) Flashcards

1
Q

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.

A

Nucleic acid extration

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2
Q

Encompass extraction of both DNA and RNA but can be more broadly characterized into chemically driven or solid phase methods.

A

Nucleic Acid Extraction (NAE) methods

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3
Q

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.

A

Chemical methods of NAE

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4
Q

Enumerate the four techniques under the chemical method of NAE

A
  1. Osmotic shock
  2. Enzymatic digestion
  3. Detergents
  4. Alkali treatment
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5
Q

The only chemical technique that the mode of lysis is HARSH

A

Alkali treatment

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6
Q

Principle of Osmotic Shock

A

Osmotic rupture of membrane

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7
Q

The cost is moderate and is usually applied for general use

A

Detergents

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8
Q

Principle of Enzymatic digestion

A

Digestion of cell wall

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9
Q

Principle of Detergents and Alkali treatment

A

Solubilization of membrane

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10
Q

Cheap at small scale; expensive at large scale

A

Enzymatic digestion

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11
Q

Used for spheroplasts and protoplasts

A

Osmotic shock

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12
Q

Used for gram-positive and gram-negative bacteria

A

Enzymatic digestion

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13
Q

The cost of these chemical techniques is CHEAP

A

Osmotic shock and alkali treatment

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14
Q

Used to extract DNA from bacteria and is based on the phenomenon of buoyant and specific density

A

Cesium Chloride (CsCl) Gradient Centrifugation with Ethidium Bromide (EtBr)

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15
Q

Principle of Ultrasonication or cavitation

A

Disruption of cells by pressure

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15
Q

Principle of Pressure cell (“French press”)

A

Disruption of cells by shear force

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15
Q

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.

A

Guanidinium Thiocyanate-Phenol-Chloroform Extraction

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16
Q

Enumerate the techniques under the Mechanical methods of NAE

A
  1. Homogenization (blade or pestle)
  2. Ultrasonication or cavitation
  3. Pressure cell (“French press”)
  4. Ball mill
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17
Q

Principle of Homogenization (blade or pestle)

A

Shredding of cells

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18
Q

Principle of Ball mill

A

Cells crushed between glass/steel/balls/beads

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19
Q

The only mechanical technique that has a MODERATE mode of lysis

A

Homogenization (blade or pestle)

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20
Q

The only mechanical technique that costs CHEAP

A

Ball mill

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21
Q

A mechanical technique that costs moderate (method of choice for large scale)

A

Homogenization (blade or pestle)

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22
Q

A mechanical technique that costs moderate to expensive

A

Ultrasonication or cavitation

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23
Q

A mechanical technique that is used for animal tissues

A

Homogenization (blade or pestle)

24
Q

To evaluate DNA purity, measure absorbance from_______ to _______ to detect other possible contaminants.

A

230nm; 320nm

24
Q

A mechanical technique that is used for Gram- negative and some and Gram positive bacteria

A

Pressure cell (“French press”)

25
Q

A mechanical technique that is good for spheroplasts but not primary cells

A

Ultrasonication or cavitation

25
Q

A mechanical technique that used for bacteria, yeast, microalgae, unicellular animal cells

A

Ball mill

26
Q

The most common purity calculation is the ratio of the absorbance at?

A

260nm divided by the reading at 280nm. (260nm/280nm)

26
Q

The most common technique to determine DNA yield and purity is measurement of?

A

Absorbance

27
Q

What are needed for the absorbance method?

A
  • Spectrophotometer equipped with a UV lamp
  • UV-transparent cuvettes (depending on the instrument)
  • Solution of purified DNA
28
Q

Where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution.

A

260nm (A260)

28
Q

Absorbance readings are performed at?

A

260nm (A260)

29
Q

Good-quality DNA will have an A260/A280 ratio of?

A

1.7-2.0

30
Q

A reading of ____ does not render the DNA unsuitable for any application, but Iower ratios indicate more contaminants are present.

A

1.6

30
Q

DNA purity formula

A

A260/A280

31
Q

Formula used for correcting for turbidity

A

DNA purity (A260/A280)= (A260 reading — A320 reading) / (A280 reading — A320 reading)

32
Q

Can indicate that organic compounds or chaotropic salts are present in the purified DNA.

A

Strong absorbance around 230nm

33
Q

This ratio can help evaluate the level of salt carryover in the purified DNA.

A

ratio of 260nm to 230nm

34
Q

In terms of absorbance, the _____the ratio, the _____ the amount of thiocyanate salt is present.

A

lower; greater

35
Q

The A260/A230 is best if greater than?

A

1.5

36
Q

A reading at ____ will indicate if there is turbidity in the solution, another indication of possible contamination.

A

320nm

37
Q

One optical density unit (or absorbance unit) at 260 nm is equivalent to?

A

50 mg/L (or 50ug/mL) of double-stranded DNA and 40 ug/mL of RNA.

38
Q

Formula for DNA Concentration (ug/mL)

A

(A260 reading — A320 reading) x dilution factor x 50ug/ml

39
Q

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.

A
40
Q

Formula for RNA Concentration (ug/mL)

A

(A260 reading — A320 reading) x dilution factor x 40ug/ml

41
Q

DNA yield (ug) formula

A

DNA concentration x total sample volume (ml)

42
Q

This is the limit for suitability in terms of purity

A

2.5

43
Q

In Guanidinium Thiocyanate-Phenol-Chloroform extraction, this can be recovered through precipitation by isopropanol and can be used for subsequent process.

A

Total RNA

44
Q

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.

A

Total RNA; DNA

44
Q

Dedicated to plasmid DNA isolation

A

Alkaline Extraction

44
Q

Used in the field of forensics for DNA extraction from various sources, such as hair, blood stain cards, and buccal swabs

A

Chelex® Extraction

44
Q

Inhibits DNA degradation by chelating metal ions which cause DNA breakdown at high temperature and lower ionic conditions

A

Chelex® Extraction

45
Q

What is the basic principle of Alkaline Extraction?

A

selective alkaline denaturation of high molecular weight chromosomal DNA, while covalently bond circular plasmid DNA remains intact.

46
Q

Alkaline Extraction involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of ______and ______ ).

A

SDS or sodium dodecyl sulfate; NaOH (sodium hyroxide)

47
Q

Poly(A) RNA hybridizes with an oligo(dT)-cellulose matrix, under high-salt conditions

A

Purification of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography

48
Q

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.

A

Solid-Phase Nucleic Acid Extraction

49
Q

Types(?) of Solid-Phase Nucleic Acid Extraction

A
  • normal/regular SPE
  • reverse SPE
  • ion exchange SPE
50
Q

Enumerate the solid phases of Solid-Phase Nucleic Acid Extraction use:

A
  • silica matrices
  • Glass Particles
  • Diatomaceous Earth
  • Magnetic Beads-Based Nucleic Acid Purification (most common)
  • Anion Exchange Material
  • Cellulose Matrix

SECOND SEM 3rd YEAR (38 decks)

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