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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
For Alkaline Lysis Method: what buffer is used
NaOH buffer
25
What is the reagent used for the digestion method in extracting DNA from hair
dithiothreitol
26
Digestion method (DNA extraction from hair) How long is the sample incubating for and at what temperature
2 hours at 56 degrees celsius
27
What is the buffer used in DNA extraction for saliva
lysis buffer | lysisbuffer: tris EDTA, SDS, Proteinase K
28
In DNA extraction from saliva How long is the sample incubating for and at what temperature
1-3 hours at 56 degrees celsius
29
What is the reagent used in DNA extraction from urine
lysis buffer
30
DNA extraction from urine: what is the first step
centrifugation
31
DNA extraction from urine: What do you discard in the second step (after centrifugation)
supernatant
32
DNA extraction from urine: how long do you incubate the sample and at what temperature
15 mins at -20 degrees celsius
33
DNA extraction from urine: last step
add lysis buffer
34
T or F bacteria and fungi 's cell walls must be broken to release the nucleic acid
T
35
DNA extraction for microorganisms What are the reagents used in the chemical method
Detergents Cetyltrimethylammonium bromide (CTAB)
36
DNA extraction for microorganisms What are the enzymes that may be used
Lysozyme and Lyticase
37
What are the two methods of DNA extraction for Formalin-Fixed, Paraffin-embedded tissue
deparaffinization Glycine in alkaline environment
38
What is the first step in deparaffinization
de-waxing
39
use this card to familiarize yourself with the steps in deparaffinization
1. De-waxing 2. heating 3. Application of organic solvents, Microwaving, heating with mineral oil 4. Digestion using proteinase K 5. purification
40
how are cell debris and partially digested organelles pelleted to make a reasonably clear supernatant
centrifugation
41
what are the commonly used procedures for purification of DNA from cell extract
phenol-chloroform extraction / organic extraction inorganic DNA extraction minicolumn purification
42
Use this card to familiarize yourself with organic extraction reagents
cell lysis buffer EDTA Proteinase K Phenol / chloroform TE buffer Ethanol
43
Organic extraction reagent: it is non ionic and has detergent salt
cell lysis buffer
44
Organic extraction reagent: breaks down polypeptides
proteinase k
45
Organic extraction reagent: keeps solution at defined pH
TE buffer
46
Organic extraction reagent: precipitates the DNA
ETHANOL
47
These are three reagents that can be used to increase DNA extraction yield after using organic extraction reagents
ribonuclease chloroform 0.3 M Na acetate in ice cold 100% ethanol
48
what is used in inorganic DNA extraction
high salt concentration solution
49
what is the effect of inadequately removed salt in inorganic DNA extraction
banding shifts
50
inorganic DNA extraction: how long is the sample incubated with proteinase K / Tris EDTA / SDS and at what temperature
56 degrees Celsius for 1-3 hours
51
inorganic DNA extraction: what reagents may be used to wash the DNA sediments
100 % ethanol or isopropanol
52
Where are the nucleic acids binded to in minicolumn purification
silica beads
53
minicolumn precipitation: what extraction method is used
spin column using a silica based extraction method
54
Enzymes, Reagents, Chemicals: Degrades single stranded RNA
RNAse (thermofisher)
55
Enzymes, Reagents, Chemicals: dissolves RNAse
buffer 1
56
Enzymes, Reagents, Chemicals: Lyse gram-negative bacteria cell wall
lysozyme (sigma)
57
Enzymes, Reagents, Chemicals: lyse gram-positive bacterial cell wall
achromopeptidase (sigma)
58
Enzymes, Reagents, Chemicals: solubilize cell membrane lipids
sodium dodecyl sulfate (SDS)
59
Enzymes, Reagents, Chemicals: digests proteins
Proteinase K (ThermoFisher)
60
Enzymes, Reagents, Chemicals: separates DNA from other cellular materials
phenol: chloroform: isoanyl alcohol
61
Enzymes, Reagents, Chemicals: precipitates DNA from solution
ethanol
62
Enzymes, Reagents, Chemicals: Dissolves precipitated and dried DNA
Tris-EDTA
63
What is the most frequently used method in concentrating DNA samples
ethanol precipitation
64
Concentration of DNA samples: what can be used to pull out adhering DNA strands in a concentrated solution
glass rod
65
Concentration of DNA samples: how can precipitated DNA be collected in dilute solutions
centrifugation and redissolving in water
66
How is the purity of DNA concentration measured
UV absorbance spectrometry
67
T or F the amount of UV radiation absorbed by a solution of DNA is indirectly proportional to the amount of DNA sample
F (directly proportional)
68
What is the ratio of absorbance of a pure DNA sample at 260 and 280 nm?
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
Measurement of purity of DNA concentration: less than 1.8
contaminated
70
Measurement of purity of DNA concentration: more than 1.8
has RNA
71
T or F RNA isolation is more sensitive and difficult to isolate than DNA
t
72
This is used in all steps of protein synthesis in all living cells and carries the genetic information for many viruses
RNA
73
What complicates RNA isolation
ribonucleases (RNAses)
74
What are the applications of RNA isolation
northern blot analysis molecular cloning in vitro translation
75
this is a common lab contaminant
RNAses
76
What are the three inhibitors of RNAse
DEPC: diethylpyrocarbonate Vanadyl ribonucleoside complexes Protein inhibitors of RNAses
77
Inhibitors of RNAses: alkylating agent, modifying proteins and nucleic acids
DEPC: diethylpyrocarbonate
78
Inhibitors of RNAses: competitive inhibitors of RNAse
Vanadyl ribonucleoside complexes
79
Inhibitors of RNAses: horse-shoe shaped, leucine rich protein, found in the cytoplasm of most mammalian tissue
protein inhibitors of RNAses
80
use this card to familiarize your self with the precautions for working with RNA in the clinical lab
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
T or F To avoid contaminated solutions / buffers, solutions must be treated with vanadyl ribonucleoside complexes
F (DEPC)
82
T or F To avoid contaminated solutions, make small batches only
T
83
What are the two steps to avoid contaminated equipments
use RNA-only pipets and glassware maintain a separate area for RNA work
84
Use this card to familiarize your self with the basic steps in isolating RNA from clinical specimens
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
Organic extraction method: disrupts cells, solubilizes cell components but maintains the integrity of RNA
Phenol / guanidium solution
86
# RNA Isolation Organic extraction method: what are the 3 phases of separation | after chloroform and centrifugation
lower organic phase middle phase w/ denatured proteins, DNA upper aqueous phase that contains RNA
87
Organic extraction method: how is RNA collected
by alcohol precipitation
88
A single step process in RNA isolation that maintains RNA integrity during tissue homogenization, while disrupting and breaking down cells and its components
TRIZOL RNA isolation protocol
89
T or F TRIZOL RNA isolation protocol is light sensitive and must be stored in an amber bottle
T
90
what does TRI in trizole stand for
total RNA isolation
91
Inorganic salt precipitation: what denatures proteins
by detergents in the presence of EDTA or other RNAse inhibitors
92
# RNA Isolation Inorganic salt precipitation: what precipitates proteins / DNA
high concentration of salt solution
93
# RNA Isolation Inorganic salt precipitation: what precipitates RNA
Alcohol, and is rehydrated
94
Use this card to familiarize yourself with filter- based RNA isolation
1. prep lysate 2. filter lysate in filtration column 3. bind RNA 4. Wash column 5. elute total RNA
95
this removes RNA from the membrane in filter-based RNA isolation
elution buffer
96
this lyses cells / homogenize the tissue in filter-based RNA isolation
lysate
97
Magnetic Particle methods: where does RNA bind to after lysis
paramagnetic beads
98
these are the equipment necessary for magnetic particle methods
bead purification kits magnetic particle processing systems magnetic separators tubes, colums, and flasks
99
what ratio is used to assess RNA purity
A260 /A280 ratio
100
An A260 /A280 ratio of _____ indicates highly purified RNA
2.0

Molecular Biology Lecture (17 decks)

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