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zzz - BIO107 - Cell and Molecular Biology > Lesson 3 - Nucleic Acid Isolation > Flashcards

Lesson 3 - Nucleic Acid Isolation Flashcards

1
Q

application of nucleic acid extraction

A
  1. DNA profiling
  2. molecular biotechnology
  3. phylogenetic studies
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2
Q

where is high quality used for

A
  1. restriction digestion
  2. gene cloning
  3. amplification
  4. DNA sequencing
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3
Q

Common sources of DNA

A
  1. whole blood
  2. hair
  3. sperm
  4. bones
  5. nails
  6. tissues
  7. saliva
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4
Q

What are the five general steps in nucleic acid isolation

A
  1. tissue homogenization and cell lysis
  2. denaturation and separation of other biomolecules from nucleic acid
  3. precipitation of nucleic acid from aqueous phase
  4. washing of precipitated nucleic acid
  5. drying of pellet and dissolution of dried pellet
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5
Q

mechanical method for tissue homogenization

A
  1. sonication
  2. grinding
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6
Q

refers to the process of applying sound energy to agitate particles or discontinuous fibers in a liquid

A

Sonication

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7
Q

reduce the size of materials to give a usable form or to separate their components

A

grinding

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8
Q

chemical used for cell lysis

A
  1. buffer
  2. salt
  3. detergent/surfactant
  4. denaturants
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9
Q

buffer

A

Tris-HCl

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10
Q

salt

A

NaCl

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11
Q

detergent/surfactant

A

SDS (sodium dodecyl sulfate)

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12
Q

purpose of detergent

A

emulsify phospholipids

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13
Q

denaturants

A

guanidinium

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14
Q

what does guanidinium do

A

inactivates RNases / DNases

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15
Q

used to treat other cellular components

A

enzymatic treatment

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16
Q

enzymatic treatment examples

A
  • lysozyme
  • cellulase
  • pectinase
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17
Q

chemical treatment for denaturation and separation of other biomolecules from the nucleic acid

A
  1. phenol
  2. choloroform
  3. isoamyl alcohol
  4. CTAB
  5. PVP
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18
Q

denature proteins

A

phenol

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19
Q

what does phenol denature

A

proteins

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20
Q

removes proteins and lipids

A

chloroform

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21
Q

what does chloroform remove

A

proteins and lipids

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22
Q

removes phenol and chloroform

A

isoamyl alcohol

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23
Q

what does isoamyl alcohol remove

A

phenol and chloroform

24
Q

removes polysaccharides

A

CTAB (cetyltrimethylammonium bromide)

25
Q

what does CTAB (cetyltrimethylammonium bromide) remove

A

polysaccharides

26
Q

removes polyphenols (alkaloids)

A

PVP (polyvinylpyrrolidone)

27
Q

what does PVP (polyvinylpyrrolidone) remove

A

polyphenols (alkaloids)

28
Q

used in enzymatic treatment during the denaturation and separation of other biomolecules from the nucleic acid

A
  • protease
  • proteinase
29
Q

sample will be separated by density with heavier stuff at the bottom of the tube and lighter stuff on top

A

centrifugation

30
Q

formed during centrifugation

A
  1. supernatant
  2. precipitate
31
Q

clear liquid free of precipitate located above the solid part

A

supernatant

32
Q

solid that forms at the bottom after centrifugation

A

precipitate

33
Q

monovalent cations

A
  • sodium
  • potassium
34
Q

alcohol used in the precipitation of nucleic acid from aqueous phase

A
  • ethanol (95%)
  • isopropanol (95%)
35
Q

used to wash precipitated nucleic acids

A

70% ethanol / isopropanol

36
Q

two types of dyring of pellet

A
  1. air drying
  2. vacuum drying
37
Q

where is dissolution of dried pellet done

A
  • sterilized molecular grade water or
  • TE (Tris-EDTA)
38
Q

EDTA

A

Ethylenediaminetetraacetic acid

39
Q

inactivates DNases

40
Q

what does EDTA inactivate

41
Q

after isolation,
__ treatment if DNA is isolated

42
Q

after isolation,
__ treatment if RNA is isolated

43
Q

storage of isolated DNA

A
  1. stock solution
  2. working solution
44
Q

stock solution temp.

A

-20°C to -80°C

45
Q

working solution temp.

46
Q

stock solution

A

pellet + Tris-EDTA

47
Q

how to determine purity and concentration of DNA isolation

A
  1. using DNA standard (different concentrations)
  2. UV spectrophotometer (purity)
48
Q

laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.

A

Gel electrophoresis

49
Q

To assess purity using the UV spectrophotometer, what is done?

A
  1. take readings at 260 nm - 280nm
  2. get ratio of A260/A280
  3. get ratio of A260/A230
50
Q

high purity for NA sample

A

A260/A280 = 1.8 to 2.0

51
Q

A260/A280 < 1.8 (or <1.6)

A

protein contamination

52
Q

protein contamination

A

A260/A280 < 1.8 (or <1.6)

53
Q

A260/A280 > 2.0

A

chloroform/phenol contamination

54
Q

chloroform/phenol contamination

A

A260/A280 > 2.0

55
Q

if A260/A230 < 2.0, it is contaminated with:

A
  1. carbohydrate carryover
  2. residual phenol/guanidinium
  3. other organic compounds
  4. salts

zzz - BIO107 - Cell and Molecular Biology (21 decks)

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