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MolBio > DNA and RNA extraction > Flashcards

DNA and RNA extraction Flashcards

1
Q

starting point in any molecular diagnostic kit

A

nucleic acid extraction

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2
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

NAE chemical method

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3
Q

enumerate the chemical techniques

A
  • osmotic shock
  • enzymatic digestion
  • detergents
  • alkali treatment
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4
Q

osmotic rupture of membrane

A

osmotic shock

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5
Q

principle of enzymatic digestion

A

digestion of cell wall

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6
Q

principle of detergent

A

solubilization of membranes

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7
Q

principle of alkali treatment

A

solubilization of membrane

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8
Q

mode of lysis (chemical)

A
  • osmotic shock - gentle
  • enzymatic digestion - gentle
  • detergents - gentle
  • alkali treatment - harsh
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9
Q

enzymatic digestion cost

A

cheap at small scale
expensive at large scale

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10
Q

most usual application of osmotic shock

A

spheroplasts and protoplasts

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11
Q

most usual application of enzymatic digestion

A

gram-positive and gram-negative bacteria

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12
Q

most usual application of detergents

A

general use

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13
Q

most usual application of alkali treatment

A

plasmid DNA

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14
Q

Cesium Chloride Gradient Centrifugation with Ethidium Bromide is baded on the phenomenon ____

used to ____

A

phenomenon of buoyant and specific density

used to extract DNA from bacteria

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15
Q

separation of RNA from DNA and proteins after extraction with an acidic solution

A

Guanidinium Thiocyanate-Phenol-Chloroform Extraction

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16
Q

Guanidinium Thiocyanate-Phenol-Chloroform Extraction consist mainly of?

A
  • GuSCN
  • sodium acetate
  • phenol
  • chloroform

followed by centrifugation

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17
Q

in Guanidinium Thiocyanate-Phenol-Chloroform Extraction, total RNA remains in the ____ while most of DNA and proteins part remain either in the ____

A

total RNA = upper aqueous phase

DNA and proteins = interphase or in the lower organic phase under acidic condition

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18
Q

Guanidinium Thiocyanate-Phenol-Chloroform Extraction: total RNA Is then recovered through?

A

precipitation by isopropanol

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19
Q

Chelex extraction is used in the field of?

A

forensics for DNA extraction from various sources such as:
- hair
- blood stain cards
- buccal swabs

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20
Q

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

A

Chelex extraction

21
Q

alkaline extraction is dedicated to?

A

plasmid DNA isolation

22
Q

principle of alkaline extraction

A

selective alkaline denaturation of high molecular weight chromosomal DNA, while covalently bond circular plasmid DNA remains intact. After neutralization, chromosomal DNA renatures and makes an insoluble precipitate, while plasmid DNA remains in the supernatant

23
Q

involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of SDS and NaOH)

A

alkaline extraction

24
Q

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

A

purification of Poly(A) + RNA by Olido(dT)-Cellulose chromatography

25
Q

based on the 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
- normal/regular SPE
- reverse SPE
- ion exchange SPE

26
Q

solid phases use (6)

A
  • silica matrices
  • glass particles
  • diatomaceous earth
  • magnetic beads-based nucleic acid purification
  • anion exchange material
  • cellulose matrix
27
Q

enumerate the mechanical techniques

A
  • homogenization (blade or pestle)
  • ultrasonication or cavitation
  • pressure cell “french press”
  • ball mill
28
Q

method of choice for large scale

A

homogenization

29
Q

most usual application of homogenization

A

animal tissues

30
Q

most usual application of ultrasonication or cavitation

A

good for spheroplasts but not primary cells

31
Q

most usual application of pressure cell

A

gram negative and some gram positive bacteria

32
Q

most usual application of ball mill

A

bacteria, yeast, microalgae, unicellular animal cells

33
Q

principle of homogenization

A

shredding of cells

34
Q

principle of ultrasonication or cavitation

A

disruption of cells by pressure

35
Q

principle of pressure cell

A

disruption of cells by shear force

36
Q

principle of ball mill

A

cells crushed between glass/steel balls/beads

37
Q

most common technique to determine DNA yield and purity?

A

measurement of absorbance

38
Q

needed for absorbance method

A

spectrophotometer with UV lamp, UV-transparent cuvettes, and a solution of purified DNA

39
Q

absorbance to evaluate DNA purity

A

260 - 280 nm (based ni maam abi)

260 - most common

40
Q

good quality DNA will have the ratio of?

A

1.7 - 2.0

41
Q

ratio that indicates more contaminants present

A

<1.6

42
Q

ratio indicates too much protein and is not acceptable

A

3

43
Q

ratio indicates contaminants and still acceptable

A

2-2.5

44
Q

strong absorbances around 230 nm can indicate that?

A

organic compounds or chaotropic salts are present

45
Q

the __ the ratio, the ___ the amount of thiocyanate salt is present

A

the LOWER the ratio, the GREATER the amount of thiocyanate salt is present

46
Q

absorbance best for greater than 1.5

A

260/320

47
Q

a reading at 320nm indicate?

A

turbidity in the solution and possible contamination

48
Q

DNA concentration absotbance

A

260 nm

49
Q

do not use ___ if no correction

A

A320

MolBio (10 decks)

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