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MODX > MEASUREMENT OF NUCLEIC ACIDQUALITYANDQUANTITY > Flashcards

MEASUREMENT OF NUCLEIC ACIDQUALITYANDQUANTITY Flashcards

1
Q

the agents used most frequently for visualization of bands after electrophoresis are

A

fluorescent dyes and silver stain

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2
Q

this dyes are specifically associated with nucleic acid

A

silver stain and fluorescent dye

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3
Q

the agent for visualization of bands that is frequently used and is specifically for detection of nucleic acids

A

fluorescent dyes

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4
Q

the agent for visualization of bands that is frequently used and for visualization of proteins and other protein related products

A

silver stain

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5
Q

quality is also known as

A

purity

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6
Q

3 main methods to identify sample quality and quantity of nucleic acids

A

electrophoresis
spectrophotometry
fluorometry

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7
Q

this method to identify sample quality is for the presence of the bands

A

electrophoresis

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8
Q

this method to identify sample quality is to check absorbance

A

spectrophotometry

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9
Q

this method to identify sample quality is for the absorbance that is based on light that is emitted

A

fluorometry

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10
Q

this fluorescent dye is responsible for attachment or intercalation of the base pairs of the nucleic acid to the dye

to make it easier - binding of dye to the DNA

A

eithidiun bromide

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11
Q

this fluorescent dye is specifically for electrophoresis

A

sybr green

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12
Q

this fluorescent dye is specifically for fluorometry

A

hoeschst dye

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13
Q

the appearance depends on the DNA isolated

A

electrophoresis

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14
Q

a high molecular weight genomic DNA should collect as a

A

bright band and low mobility

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15
Q

a high quality preparation of RNA will yield into two distinct band of rRNA, what are they?

A

mRNA and rRNA

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16
Q

the most numerous RNA and present in ribosomes

A

rRna

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17
Q

The RNA in ___ is considered as degraded RNA or
smeared

A

0.5kb

and if degraded, we will not be able to use it for molecular assays

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18
Q

The integrity of this bands is an indication of the integrity of the other RNA species present in the same sample

which concept is this

A

electrophoresis

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19
Q

after agarose gel electrophoresis, we can see different types of bands

A

super coild
nick
relax
linear plasmids
molecular ladders

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20
Q

a compact type of band that will travel farther in the gel

A

super coiled

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21
Q

a single stranded breaks in the bands

A

nick

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22
Q

a double stranded breaks in a band

A

relax

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23
Q

this band migrate according to the size of plasmid

A

linear plasmids

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24
Q

this band determines the size of the band

A

molecular ladders

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25
Q

most commonly used in visualizing nucleic acid

A

fluorescent dyes

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26
Q

an intercalation in visualizing nucleic acid in electrophoresis

A

fluorescent dyes

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27
Q

is a fluorescent dye that is the most widely used dye in early DNA and RNA analysis

A

ethidium bromide

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28
Q

ethidium bromide is also known as

A

3,8 diamino 5 ethyl 6 phenylphenanthridinium bromide

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29
Q

__is carcinogenic, precautions are required to
limit exp

A

EtBr

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30
Q

under excitation with UV light at 300 nm, EtBr in DNA will emit visible light at

A

590 nm

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31
Q

after soaking or running in dye, the dna illuminated with UV light will appear as what color in the gel

A

orange

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32
Q

used to visualize the image that can be capture

A

gel documentation box

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33
Q

the color of raw extracted nucleic acid is

A

colorless - will turn blue once in contact with ethidium bromide

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34
Q

MINORGROOVE–BINDING DYES used what stains

A

SYBR green

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35
Q

SYBR green is introduced on what year

A

1995

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36
Q

SYBR green that is used for double stranded DNA

A

SYBR I

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37
Q

SYBR green that is used for single stranded DNA

A

SYBR green II

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38
Q

this SYBR is used for both DNA and RNA staining

A

SYBR gold

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39
Q

SYBR green -has sensitivity of 2ng/mL. however, it is not specific for RNA, but it will bind double stranded DNA

true or false

A

true

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40
Q

difference of SYBR 1 green to EtBr

A

it does not intercalate between bases; it sits in the minor groove of the double helix

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41
Q

SYBR green in association
with DNA or RNA also emits
light in the ___range (522
nm).

A

orange

42
Q

in agarose gel electrophoresis, SYBR staining is how many times more sensitive than ethidium bromide

A

25 to 100 times more

43
Q

detection level of SYBR green compared to ETBR proving that it’s more sensitive than ETBR

A

SYBR green - 60 pg of double stranded DNA
EtBr- 5 ng

44
Q

the ability of the method to measure
the smallest concentration of particular analyte

A

sensitivity

45
Q

the smallest concentration of analyte that will give the positive result

A

limit of detection

46
Q

this dye is a cyanine dye of proprietary structure

A

SYBR gold stain

47
Q

Like SYBR green, SYBR gold is
excited by UV light (300 nm
wavelength). SYBR gold emits
light at ___ nm.

A

537

48
Q

its fluorescence increases more than 1000 fold upon binding to double or single stranded DNA or to RNA

A

SYBR gold stain

49
Q

why is EtBr still the one being use in most of laboratories despite of availability of SYBR

A

due to the requirement for the special OPTICAL FILTERS

50
Q

is the preferred dye for real time PCR methods

A

SYBR green

51
Q

another sensitive staining system that is originally developed for protein visualization is

A

silver stain

52
Q

this staining system (silver stain), after electrophoresis, the sample is fixed with

A

methanol and acetic acid

53
Q

after fixing the sample in methanol and acetic acid as part of the process of silver staining, it will then be impregnated with

A

ammoniacal silver (silver diamine) solutions or silver nitrate in a weakly acid solution

54
Q

it is especially useful for protein analysis and for detection of limiting amounts of product

A

silver stain

55
Q

sensitivity of silver stain

A

14.6 picograms

56
Q

running time of silver stain

A

6-7 mns

57
Q

reagents used in silver staining

A

silver nitrate, sodium hydroxide, formaldehyde

58
Q

more sensitive than SYBR green

A

silver stain

59
Q

the sample absorbance are determined on the spectrophotometer at what nm

A

260 nm and 280 nm

60
Q

nucleic acid absorb light at _____ nm

A

260 nm

61
Q

protein absorbs light at _____ nm

A

280 nm; 230 nm

62
Q

a260/a280 is a measure of

A

DNA purity

63
Q

It is used to detect the quality of nucleic acid and contamination of protein or organic solvents

A

spectrophotometry

64
Q

the absorbance wavelength is directly proportional to the concentration of nucleic acid

A

spectrophotometry

65
Q

in spectrophotometry, using ___ law, concentration can be determined from the absorptivity constants

A

beer-lambert law

66
Q

the absorptivity constant of DNA (optical density )

A

50

67
Q

the absorptivity constant of RNA (optical density )

A

40

68
Q

in terms of molecular bio, we are using ___ t measure the purity and concentration of nucleic acids

A

spectrophotometer

69
Q

common contaminant and their wavelength peak absorbance

230 nm

A

organic compounds

70
Q

common contaminant and their wavelength peak absorbance

270 nm

A

phenol

71
Q

common contaminant and their wavelength peak absorbance

280 nm

A

protein

72
Q

common contaminant and their wavelength peak absorbance

> 330 nm

A

particulate matter

73
Q

how to determine the Concentration

A

OD 260 nm X OD unit X dilution factor

74
Q

one optical density OD unit or absorbance unit at 260 nm is equalt to

A

50 ug/ml for dna
40 ug/ml for rna

75
Q

Concentration is usually expressed in

A

MCG
(micrograms/mL)

76
Q

The OD260nm should be __ times more than OD280nm

A

1.6-2.00

77
Q

OD260nm ÷ OD280nm ___

A

=OD Ratio

78
Q

If the OD ratio is less than __for DNA, this indicate contamination

A

1.6

usually with protein

79
Q

If the OD ratio is __for RNA, this indicate contamination

A

2-2.3

80
Q

in DNA, if the OD ration is higher than 2.0, it may be contaminated by ___

A

RNA

81
Q

Pure preparation of DNA and RNA have OD Ratio of __ respectively

A

1.8and 2.0

82
Q

a DNA preparation dulited 1:100 yields an absorbance reading of __ at 260 nm

A

0.200

83
Q

Fluorometry is also called as

A

fluorescence spectroscopy

84
Q

Fluorometry utilizes ___ dyes which specifically bind DNA or RNA

A

fluorescent dyes

85
Q

fluorometry requires what pressure and and what standard

A

negative control (to set the zero point in the fluorometer) and a standard of known concentration

86
Q

The fluorescent dyes are relatively specific
to nucleic acids as opposed to protein and other cellular components

t
or f

A

t

87
Q

The fluorescence of the dyes increases when they
bind

A

nucleic acids

88
Q

It is preferred for very low amount of nucleic acid

A

10-100 ng/mL

89
Q

fluorometry

It uses how many monochromator before it goes to detect the sample

A

two - primary and secondary

90
Q

dyes in fluorometry

A

hoechst dye 33258
pico green dye
oligreen dye

91
Q

sensitivity of Hoechst dye 33258

A

200 ng of DNA/ml

92
Q

this dye is very important for adenine, it combines adenine bands. It is very specific for impact double stranded DNA

A

Hoechst dye

93
Q

this fluorometric dye that is for double stranded DNA

A

pico green dye

94
Q

this fluorometric dye

more sensitive than hoechst

A

pico green dye

95
Q

this fluorometric dye

sensitivity of pico green dye

A

25 pg/ml

96
Q

this fluorometric dye

for single stranded DNA

A

oligreen Dye

97
Q

this fluorometric dye

sensitivity of OliGreen dye

A

100 pg/ml

98
Q

a type of cuvette that is use din spectrophotometer

A

glass aluminum silica

99
Q

a type of quarts cuvette for fluorometry

A

quartz cuvette

100
Q

advantage of fluorometry

A

about 1000 times more sensitive than spectrophotometric absorbance

less susceptible to protein and RNA contamination

101
Q

disadvantage of fluorometry

A

does not give a crude measurement of purity
does not assure that the dna or rna is not degraded
frosted part of the glass interferes with the detection of fluorescent light when using glass cuvettes in the fluorometer

102
Q

we measure the presence or absence of DNA

A

fluorometry

o No absorbance
o No concentration
o Only presence or absence

MODX (16 decks)

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