Techniques Quiz 3 Flashcards
(83 cards)
1
Q
Integrity of RNA can be assessed by…
A
Fluorometric spectroscopy
UV spectroscopy
Analytical agarose electrophoresis
2
Q
What animal were tests run on?
A
Mus musculus
3
Q
Estimated gene count of mice
A
23,700
4
Q
Estimated gene count of humans
A
21,306
5
Q
Human brain mass
A
1300g
6
Q
Mouse brain mass
A
400mg
7
Q
Brain/body ratio for mice and humans
A
1/40
8
Q
How much brain did each student get?
A
~100mg
9
Q
How much liver did each student get?
A
~200mg
10
Q
What was the goal of RiboNucleic Acid Isolation
A
Total RNA
11
Q
How much of the total RNA was messenger RNA?
A
3%
12
Q
How much of the total RNA was ncRNA
A
97%
13
Q
How much of the total RNA is ribosomal RNA (rRNA)
A
80%
14
Q
RNA that codes for proteins
A
mRNA
15
Q
RNA used in translation
A
rRNA
16
Q
Make up the large subunit of the ribosome
A
28S, 5.8S, 5S
17
Q
Make up the small subunit of the ribosome
A
18S
18
Q
80nt; used in translation
A
tRNA
19
Q
Used in the splicing of pre-mRNA
A
snRNA
20
Q
Handles nucleotide modifications of tRNAs, rRNAs, and snRNAs with associated enzymes
A
snoRNA
21
Q
Used in RNA processing, DNA replication, and target of autoantibodies in lupus
A
Y RNA
22
Q
Used in telomere synthesis
A
Telomerase RNA
23
Q
Used in transcriptional attenuation, mRNA degradation, mRNA stabilization, and translation block
A
antisense RNA
24
Q
Handles gene regulation via binding to complementary mRNA
A
miRNA
25
Transposon defense via RNA/protein interaction - transcription silencing
piRNA
26
>200nt long
Long ncRNA
27
Cleaves phosphodiester bonds in the middle of ribonucleic acids
Endoribonuclease
28
Sequence specific ribonuclease for ssRNA
RNase A
29
Ribonuclease that cleaves RNA of RNA/DNA duplex
RNase H
30
3'-5' maturation of many ssRNAs
RNase T
31
3'-5' processing of pre-tRNAs
RNase D
32
What group cleaves the RNA bonds?
Imidazole group
33
Reacts with the imidazole ring of RNases to inactivate activity
Diethylpyrocarbonate (DEPC)
34
What is a large source of RNase
The human body
35
How much of what solution was used to preserve the tissues after extraction?
1mL of RNALater
36
Which tissue underwent organic extraction?
Liver tissue
37
What solution was added to liver tissue and how much per 100mg?
1000uL of Tri-Reagent
38
What tissue underwent spin-column extraction?
Brain tissue
39
What solution was added to brain tissue and how much per 30mg?
600uL of lysis buffer
40
What solution was added after the lysis buffer and how much per 1mL of buffer?
10uL of betamercaptoethanol (B-ME)
41
Used for tissue homogenization and disruption of RNases in RNA isolation
Tri-reagent
42
Method that results in high purity and high efficiency removal of contaminants from small RNA species
Phenol-chloroform extractions (organic)
43
What function does phenol to in phenol-chloroform extractions
Dissolves proteins
44
What function does chloroform do in phenol-chloroform extractions?
Dissolves lipids
45
What function does isoamyl alcohol do in phenol-chloroform extractions?
Reduces foaming and inactivates RNase
46
Found in the aqueous phase of phenol-chloroform extractions
RNA
47
Found in the interphase phase of phenol-chloroform extractions
Proteins
48
Found in the organic phase of phenol-chloroform extractions
DNA
49
What wavelengths is RNA indicated at?
260nm and 280nm
50
Filter-based isolation system that captures RNA in a silica-based mesh resulting in high purity RNA
Spin-column isolation
51
Allows negative RNA to bind to silicate filter via NA+ interactions
High salt buffer
52
Made up of Tris and EDTA
TE Buffer
53
Used to wash the sample of loose binding contaminants
Higher stringency washes
54
Higher stringency washes have ___________
Lower salt concentrations
55
Spin column homogenate is in ________ ___________
Guanidinium isothiocyanate
56
Lower salt buffers used for elution cause RNA to lose grip on matrix by ________ the nucleic acid structure
Hydrating
57
Neutralizes charges on sugar phosphate backbone
Salt
58
Common salts used in nucleic acid precipitation
1. S_______ A______
2. S_______ C______
3. L_______ C______
4. A_______ A______
Sodium acetate
Sodium chloride
Lithium chloride
Ammonium acetate
59
Routine salt in RNA preps and RNA
Sodium acetate
60
Salt used in DNA samples containing SDS - keeps SDS soluble in 70% ethanol
Sodium chloride
61
Used in RNA preps and more soluble than sodium acetate in ethanol.
Chloride ions will disrupt activity of DNA polymerase. Not to use if doing RT
Lithium chloride
62
Used in DNA and RNA preps but cation can inhibit some downstream enzymes
Ammonium acetate
63
Defined as a set of conditions that are used to control the "strandedness" of nucleic acids
Stringency
64
Things that can affect the strandedness of nucleic acids
1. Degree of complementation
2. Salt concentration of the buffer
3. Temperature
65
Results in lower stringency
Higher salt concentrations and lower temps
66
Results in higher stringency
Lower salt concentrations and higher temps
67
More non-specific binding can occur
Much more forgiving on what can bind
Lower stringency
68
Less specific interactions will come free, leaving only the most highly complementary pairing in place
Higher stringency
69
Represents the charge of each ion in Coulomb's Law
Q1 and Q2
70
Represents the distance between each ion in Coulomb's Law
r
71
Represents the dielectric constant / measure of polarity in Coulomb's Law
E
72
Represents the electrostatic force / attraction between ions in Coulomb's Law
F
73
Prevents ions from forming ionic compounds decreasing the electrostatic force
Hydration shells
74
What should the ratio of 28S:18S be?
2:1
75
How many bp is 28S rRNA?
4700bp
76
How many bp is 18S rRNA?
1900bp
77
How many bp is 5/5.8S rRNA
~300bp
78
Percentage of TAE gel used
1.2%
79
How many mL of 1x TAE were used?
30mL
80
Dilution of SYBR Safe used
1:10000
81
Construction of RNA gel sample
2uL DNA
2uL 6X Loading Dye
8uL Water
10uL Loaded
82
What do nucleic acids need to be assessed for?
1. C___________
2. P_____
3. I________
1. Concentration
2. Purity
3. Integrity
83
Coulomb's Law equation
F = (Q1 x Q2)/(E x (r^2))
