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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
most commonly used in visualizing nucleic acid
fluorescent dyes
26
an intercalation in visualizing nucleic acid in electrophoresis
fluorescent dyes
27
is a fluorescent dye that is the most widely used dye in early DNA and RNA analysis
ethidium bromide
28
ethidium bromide is also known as
3,8 diamino 5 ethyl 6 phenylphenanthridinium bromide
29
__is carcinogenic, precautions are required to limit exp
EtBr
30
under excitation with UV light at 300 nm, EtBr in DNA will emit visible light at
590 nm
31
after soaking or running in dye, the dna illuminated with UV light will appear as what color in the gel
orange
32
used to visualize the image that can be capture
gel documentation box
33
the color of raw extracted nucleic acid is
colorless - will turn blue once in contact with ethidium bromide
34
MINORGROOVE–BINDING DYES used what stains
SYBR green
35
SYBR green is introduced on what year
1995
36
SYBR green that is used for double stranded DNA
SYBR I
37
SYBR green that is used for single stranded DNA
SYBR green II
38
this SYBR is used for both DNA and RNA staining
SYBR gold
39
SYBR green -has sensitivity of 2ng/mL. however, it is not specific for RNA, but it will bind double stranded DNA true or false
true
40
difference of SYBR 1 green to EtBr
it does not intercalate between bases; it sits in the minor groove of the double helix
41
SYBR green in association with DNA or RNA also emits light in the ___range (522 nm).
orange
42
in agarose gel electrophoresis, SYBR staining is how many times more sensitive than ethidium bromide
25 to 100 times more
43
detection level of SYBR green compared to ETBR proving that it's more sensitive than ETBR
SYBR green - 60 pg of double stranded DNA EtBr- 5 ng
44
the ability of the method to measure the smallest concentration of particular analyte
sensitivity
45
the smallest concentration of analyte that will give the positive result
limit of detection
46
this dye is a cyanine dye of proprietary structure
SYBR gold stain
47
Like SYBR green, SYBR gold is excited by UV light (300 nm wavelength). SYBR gold emits light at ___ nm.
537
48
its fluorescence increases more than 1000 fold upon binding to double or single stranded DNA or to RNA
SYBR gold stain
49
why is EtBr still the one being use in most of laboratories despite of availability of SYBR
due to the requirement for the special OPTICAL FILTERS
50
is the preferred dye for real time PCR methods
SYBR green
51
another sensitive staining system that is originally developed for protein visualization is
silver stain
52
this staining system (silver stain), after electrophoresis, the sample is fixed with
methanol and acetic acid
53
after fixing the sample in methanol and acetic acid as part of the process of silver staining, it will then be impregnated with
ammoniacal silver (silver diamine) solutions or silver nitrate in a weakly acid solution
54
it is especially useful for protein analysis and for detection of limiting amounts of product
silver stain
55
sensitivity of silver stain
14.6 picograms
56
running time of silver stain
6-7 mns
57
reagents used in silver staining
silver nitrate, sodium hydroxide, formaldehyde
58
more sensitive than SYBR green
silver stain
59
the sample absorbance are determined on the spectrophotometer at what nm
260 nm and 280 nm
60
nucleic acid absorb light at _____ nm
260 nm
61
protein absorbs light at _____ nm
280 nm; 230 nm
62
a260/a280 is a measure of
DNA purity
63
It is used to detect the quality of nucleic acid and contamination of protein or organic solvents
spectrophotometry
64
the absorbance wavelength is directly proportional to the concentration of nucleic acid
spectrophotometry
65
in spectrophotometry, using ___ law, concentration can be determined from the absorptivity constants
beer-lambert law
66
the absorptivity constant of DNA (optical density )
50
67
the absorptivity constant of RNA (optical density )
40
68
in terms of molecular bio, we are using ___ t measure the purity and concentration of nucleic acids
spectrophotometer
69
common contaminant and their wavelength peak absorbance 230 nm
organic compounds
70
common contaminant and their wavelength peak absorbance 270 nm
phenol
71
common contaminant and their wavelength peak absorbance 280 nm
protein
72
common contaminant and their wavelength peak absorbance >330 nm
particulate matter
73
how to determine the Concentration
OD 260 nm X OD unit X dilution factor
74
one optical density OD unit or absorbance unit at 260 nm is equalt to
50 ug/ml for dna 40 ug/ml for rna
75
Concentration is usually expressed in
MCG (micrograms/mL)
76
The OD260nm should be __ times more than OD280nm
1.6-2.00
77
OD260nm ÷ OD280nm ___
=OD Ratio
78
If the OD ratio is less than __for DNA, this indicate contamination
1.6 usually with protein
79
If the OD ratio is __for RNA, this indicate contamination
2-2.3
80
in DNA, if the OD ration is higher than 2.0, it may be contaminated by ___
RNA
81
Pure preparation of DNA and RNA have OD Ratio of __ respectively
1.8and 2.0
82
a DNA preparation dulited 1:100 yields an absorbance reading of __ at 260 nm
0.200
83
Fluorometry is also called as
fluorescence spectroscopy
84
Fluorometry utilizes ___ dyes which specifically bind DNA or RNA
fluorescent dyes
85
fluorometry requires what pressure and and what standard
negative control (to set the zero point in the fluorometer) and a standard of known concentration
86
The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components t or f
t
87
The fluorescence of the dyes increases when they bind
nucleic acids
88
It is preferred for very low amount of nucleic acid
10-100 ng/mL
89
fluorometry It uses how many monochromator before it goes to detect the sample
two - primary and secondary
90
dyes in fluorometry
hoechst dye 33258 pico green dye oligreen dye
91
sensitivity of Hoechst dye 33258
200 ng of DNA/ml
92
this dye is very important for adenine, it combines adenine bands. It is very specific for impact double stranded DNA
Hoechst dye
93
this fluorometric dye that is for double stranded DNA
pico green dye
94
this fluorometric dye more sensitive than hoechst
pico green dye
95
this fluorometric dye sensitivity of pico green dye
25 pg/ml
96
this fluorometric dye for single stranded DNA
oligreen Dye
97
this fluorometric dye sensitivity of OliGreen dye
100 pg/ml
98
a type of cuvette that is use din spectrophotometer
glass aluminum silica
99
a type of quarts cuvette for fluorometry
quartz cuvette
100
advantage of fluorometry
about 1000 times more sensitive than spectrophotometric absorbance less susceptible to protein and RNA contamination
101
disadvantage of fluorometry
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
we measure the presence or absence of DNA
fluorometry o No absorbance o No concentration o Only presence or absence

MODX (17 decks)

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