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Molecular Biology Lecture > 3: DNA and RNA Extraction (PRELIMS) lec based > Flashcards

3: DNA and RNA Extraction (PRELIMS) lec based Flashcards

1
Q

a procedure used to isolate DNA from the nucleus of cells from other cellular components

A

DNA Extraction

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2
Q

two main methods of cell lysis in DNA purification

A

physical and chemical methods

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3
Q

What is the purpose of DNA extraction

A

to obtain DNA in a relatively purified from that can be used for further investigations

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4
Q

T or F

DNA can only be isolated from a living organism

A

F (living or dead organism)

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5
Q

use this card to familiarize yourself with sources of DNA

A

whole blood
hair
sperm
bones
nails
tissues
saliva
buccal swabs
epithelial cells
urine

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6
Q

use this card to familiarize your self with the steps in DNA extraction (or enumerate it if ur a masochist)

A
  1. preparation of a cell extract
  2. purification of DNA from a cell extract
  3. concentration of DNA samples
  4. Measurement of purity of the DNA concentration
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7
Q

What is the other term for cell extract preparation

A

cell lysis

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8
Q

what does EDTA remove to compromise the stability of the cell membrane?

A

Calcium and Mg ions

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8
Q

what is the main purpose of cell lysis

A

liberate / expose the DNA

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9
Q

what does the SDS (sodium dodecyl sulfate) remove to disrupt the cell membrane

A

lipids of the cell membrane

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10
Q

What method is used to extract DNA from a whole blood sample

A

Ficoll-directed gradient through centrifugation
Proteinase K
Phenol

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11
Q

what reagent is used in Ficoll-directed gradient through centrifugation

A

Ficoll-paque reagent

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12
Q

T or F

In using the Ficoll-Paque reagent, DNA is found at the top most layer after centrifugation

A

F (at the bottom)

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13
Q

Whole blood

what is proteinase K mixed with for an enzymatic method of DNA extraction

A

whole blood + Tris EDTA + SDS + MgCl

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14
Q

what is phenol mixed with for DNA extraction in whole blood

A

whole blood + Tris HCl + phenol + Mg

process for 4 hours

centrifuge

wala na ata mg here (edit)

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15
Q

At what room temperature are Dry blood spots stored at?

A

room temp or -20 degrees Celsius for prolonged storage

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16
Q

what are the two reagents used to extract DNA from dry blood spots

A

Chelex-100 and Insta Gene Matrix

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17
Q

at what temperature is the sample with chelex reagent left to incubate in?

A

100 degrees (boiling)

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18
Q

at what temperature is the sample with InstaGene Matrix left to incubate in ?

A

56 degrees celsius

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19
Q

how long is the sample with chelex left to incubate

A

8 mins

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20
Q

how long is the sample with instagene left to incubate

A

30 mins

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21
Q

Where is the DNA found in both chelex and instagene methods after centrifugation

A

DNA will be found at the supernatant

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22
Q

what are the two methods of extracting DNA from hair

A

alkaline lysis method and digestion

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23
Q

For alkaline Lysis Method:

how long is the sample incubating for and at what temperature

A

95 degrees Celsius for 10 mins

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24
Q

For Alkaline Lysis Method:

what buffer is used

A

NaOH buffer

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25
Q

What is the reagent used for the digestion method in extracting DNA from hair

A

dithiothreitol

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26
Q

Digestion method (DNA extraction from hair)

How long is the sample incubating for and at what temperature

A

2 hours at 56 degrees celsius

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27
Q

What is the buffer used in DNA extraction for saliva

A

lysis buffer

lysisbuffer: tris EDTA, SDS, Proteinase K

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28
Q

In DNA extraction from saliva

How long is the sample incubating for and at what temperature

A

1-3 hours at 56 degrees celsius

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29
Q

What is the reagent used in DNA extraction from urine

A

lysis buffer

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30
Q

DNA extraction from urine:
what is the first step

A

centrifugation

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31
Q

DNA extraction from urine:

What do you discard in the second step (after centrifugation)

A

supernatant

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32
Q

DNA extraction from urine:

how long do you incubate the sample and at what temperature

A

15 mins at -20 degrees celsius

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33
Q

DNA extraction from urine:

last step

A

add lysis buffer

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34
Q

T or F

bacteria and fungi ‘s cell walls must be broken to release the nucleic acid

A

T

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35
Q

DNA extraction for microorganisms

What are the reagents used in the chemical method

A

Detergents
Cetyltrimethylammonium bromide (CTAB)

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36
Q

DNA extraction for microorganisms

What are the enzymes that may be used

A

Lysozyme and Lyticase

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37
Q

What are the two methods of DNA extraction for Formalin-Fixed, Paraffin-embedded tissue

A

deparaffinization
Glycine in alkaline environment

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38
Q

What is the first step in deparaffinization

A

de-waxing

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39
Q

use this card to familiarize yourself with the steps in deparaffinization

A
  1. De-waxing
  2. heating
  3. Application of organic solvents, Microwaving, heating with mineral oil
  4. Digestion using proteinase K
  5. purification
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40
Q

how are cell debris and partially digested organelles pelleted to make a reasonably clear supernatant

A

centrifugation

41
Q

what are the commonly used procedures for purification of DNA from cell extract

A

phenol-chloroform extraction / organic extraction
inorganic DNA extraction
minicolumn purification

42
Q

Use this card to familiarize yourself with organic extraction reagents

A

cell lysis buffer
EDTA
Proteinase K
Phenol / chloroform
TE buffer
Ethanol

43
Q

Organic extraction reagent:

it is non ionic and has detergent salt

A

cell lysis buffer

44
Q

Organic extraction reagent:

breaks down polypeptides

A

proteinase k

45
Q

Organic extraction reagent:

keeps solution at defined pH

46
Q

Organic extraction reagent:

precipitates the DNA

47
Q

These are three reagents that can be used to increase DNA extraction yield after using organic extraction reagents

A

ribonuclease
chloroform
0.3 M Na acetate in ice cold 100% ethanol

48
Q

what is used in inorganic DNA extraction

A

high salt concentration solution

49
Q

what is the effect of inadequately removed salt in inorganic DNA extraction

A

banding shifts

50
Q

inorganic DNA extraction:

how long is the sample incubated with proteinase K / Tris EDTA / SDS and at what temperature

A

56 degrees Celsius for 1-3 hours

51
Q

inorganic DNA extraction:

what reagents may be used to wash the DNA sediments

A

100 % ethanol or isopropanol

52
Q

Where are the nucleic acids binded to in minicolumn purification

A

silica beads

53
Q

minicolumn precipitation:

what extraction method is used

A

spin column using a silica based extraction method

54
Q

Enzymes, Reagents, Chemicals:

Degrades single stranded RNA

A

RNAse (thermofisher)

55
Q

Enzymes, Reagents, Chemicals:

dissolves RNAse

56
Q

Enzymes, Reagents, Chemicals:

Lyse gram-negative bacteria cell wall

A

lysozyme (sigma)

57
Q

Enzymes, Reagents, Chemicals:

lyse gram-positive bacterial cell wall

A

achromopeptidase (sigma)

58
Q

Enzymes, Reagents, Chemicals:

solubilize cell membrane lipids

A

sodium dodecyl sulfate (SDS)

59
Q

Enzymes, Reagents, Chemicals:
digests proteins

A

Proteinase K (ThermoFisher)

60
Q

Enzymes, Reagents, Chemicals:

separates DNA from other cellular materials

A

phenol: chloroform: isoanyl alcohol

61
Q

Enzymes, Reagents, Chemicals:

precipitates DNA from solution

62
Q

Enzymes, Reagents, Chemicals:

Dissolves precipitated and dried DNA

63
Q

What is the most frequently used method in concentrating DNA samples

A

ethanol precipitation

64
Q

Concentration of DNA samples:

what can be used to pull out adhering DNA strands in a concentrated solution

65
Q

Concentration of DNA samples:

how can precipitated DNA be collected in dilute solutions

A

centrifugation and redissolving in water

66
Q

How is the purity of DNA concentration measured

A

UV absorbance spectrometry

67
Q

T or F

the amount of UV radiation absorbed by a solution of DNA is indirectly proportional to the amount of DNA sample

A

F (directly proportional)

68
Q

What is the ratio of absorbance of a pure DNA sample at 260 and 280 nm?

A

1.8

(di ko gates yung phrasing pwede po pa explain sakin ehe basta yan ang nasa ppt; 1.8 daw yung ratio of absorbance bb)

69
Q

Measurement of purity of DNA concentration:

less than 1.8

A

contaminated

70
Q

Measurement of purity of DNA concentration:

more than 1.8

71
Q

T or F

RNA isolation is more sensitive and difficult to isolate than DNA

72
Q

This is used in all steps of protein synthesis in all living cells and carries the genetic information for many viruses

73
Q

What complicates RNA isolation

A

ribonucleases (RNAses)

74
Q

What are the applications of RNA isolation

A

northern blot analysis
molecular cloning
in vitro translation

75
Q

this is a common lab contaminant

76
Q

What are the three inhibitors of RNAse

A

DEPC: diethylpyrocarbonate
Vanadyl ribonucleoside complexes
Protein inhibitors of RNAses

77
Q

Inhibitors of RNAses:

alkylating agent, modifying proteins and nucleic acids

A

DEPC: diethylpyrocarbonate

78
Q

Inhibitors of RNAses:

competitive inhibitors of RNAse

A

Vanadyl ribonucleoside complexes

79
Q

Inhibitors of RNAses:

horse-shoe shaped, leucine rich protein, found in the cytoplasm of most mammalian tissue

A

protein inhibitors of RNAses

80
Q

use this card to familiarize your self with the precautions for working with RNA in the clinical lab

A
  1. use RNAse-free tubes and pipet tips
  2. always wear gloves and work in a hood
  3. treat liquids with DEPC
  4. cells or tissues must be rapidly and efficiently disrupted
  5. Denature of nucleic acid-protein complexes
  6. RNA selectively partitioned from DNA and protein
81
Q

T or F

To avoid contaminated solutions / buffers, solutions must be treated with vanadyl ribonucleoside complexes

82
Q

T or F

To avoid contaminated solutions, make small batches only

83
Q

What are the two steps to avoid contaminated equipments

A

use RNA-only pipets and glassware

maintain a separate area for RNA work

84
Q

Use this card to familiarize your self with the basic steps in isolating RNA from clinical specimens

A
  1. cell lysis
  2. denature / digest proteins
  3. separate proteins, DNA and contamination from RNA
  4. Precipitate RNA if necessary
  5. resuspend RNA in final buffer
85
Q

Organic extraction method:

disrupts cells, solubilizes cell components but maintains the integrity of RNA

A

Phenol / guanidium solution

86
Q

RNA Isolation

Organic extraction method:

what are the 3 phases of separation

after chloroform and centrifugation

A

lower organic phase
middle phase w/ denatured proteins, DNA
upper aqueous phase that contains RNA

87
Q

Organic extraction method:

how is RNA collected

A

by alcohol precipitation

88
Q

A single step process in RNA isolation that maintains RNA integrity during tissue homogenization, while disrupting and breaking down cells and its components

A

TRIZOL RNA isolation protocol

89
Q

T or F

TRIZOL RNA isolation protocol is light sensitive and must be stored in an amber bottle

90
Q

what does TRI in trizole stand for

A

total RNA isolation

91
Q

Inorganic salt precipitation:

what denatures proteins

A

by detergents in the presence of EDTA or other RNAse inhibitors

92
Q

RNA Isolation

Inorganic salt precipitation:

what precipitates proteins / DNA

A

high concentration of salt solution

93
Q

RNA Isolation

Inorganic salt precipitation:

what precipitates RNA

A

Alcohol, and is rehydrated

94
Q

Use this card to familiarize yourself with filter- based RNA isolation

A
  1. prep lysate
  2. filter lysate in filtration column
  3. bind RNA
  4. Wash column
  5. elute total RNA
95
Q

this removes RNA from the membrane in filter-based RNA isolation

A

elution buffer

96
Q

this lyses cells / homogenize the tissue in filter-based RNA isolation

97
Q

Magnetic Particle methods:

where does RNA bind to after lysis

A

paramagnetic beads

98
Q

these are the equipment necessary for magnetic particle methods

A

bead purification kits
magnetic particle processing systems
magnetic separators
tubes, colums, and flasks

99
Q

what ratio is used to assess RNA purity

A

A260 /A280 ratio

100
Q

An A260 /A280 ratio of _____ indicates highly purified RNA

Molecular Biology Lecture (17 decks)

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