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5-8 NA Isolation Flashcards
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5-8 NA Isolation Flashcards
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1
Question
What is Chelex resin used for in DNA extraction?
Answer
Chelex resin is used for simple extraction of DNA from minimal samples by chelating cations.
2
Question
Describe the process of using Chelex resin in DNA extraction.
Answer
A suspension of 10 Chelex resin beads is mixed with the specimen, cells are lysed by boiling, and the sample is then centrifuged, with DNA in the supernatant that may be further extracted with chloroform before use in amplification procedures.
3
Question
Why is the Chelex resin method commonly used in forensic applications?
Answer
The Chelex resin method is commonly used in forensic applications due to its simplicity, safety compared to phenol, and effectiveness in extracting DNA from minimal samples.
4
Question
What is the advantage of using Chelex resin for DNA isolation when only a minimal sample is available?
Answer
The advantage of using Chelex resin for DNA isolation with a minimal sample is that it is suitable for performing amplification procedures or PCR with very small amounts of sample.
5
Question
How is mitochondrial DNA isolated using centrifugation?
Answer
Mitochondrial DNA is isolated by homogenizing the sample, centrifuging at low speed to pellet cell debris, centrifuging at high speed to pellet mitochondria, and lysing with detergent and proteinase.
6
Question
What is Chelex used for in molecular biology procedures like amplification or PCR?
Answer
Chelex can be used when a very small amount of sample is available for DNA isolation.
7
Question
Describe the steps involved in the isolation of mitochondrial DNA.
Answer
1. Homogenize the sample by grinding on ice. 2. Centrifuge the homogenate at low speed to pellet cell debris and intact cells. 3. Centrifuge the supernatant at high speed to pellet mitochondria. 4. Lysed with detergent (SDS, Triton X) and treated with proteinase K. 5. Precipitate with cold ethanol and resuspend in water/buffer.
8
Question
What is the importance of being cautious about RNAse contamination in the lab during RNA isolation?
Answer
RNAse is a common laboratory contaminant that can degrade RNA samples. RNases can renature and regain activity, even after autoclaving. It is important to use RNAse-free materials and follow strict protocols to prevent contamination.
9
Question
Why do we have to be cautious in the lab when working with mRNA?
Answer
Due to the presence of RNase, a common laboratory contaminant that can degrade RNA molecules.
10
Question
What is RNAse?
Answer
Small proteins that can renature and regain activity even after autoclaving, and can be active at temperatures as low as 20C.
11
Question
What is the importance of using RNase-free materials in the lab?
Answer
To avoid contamination and ensure that RNases are eliminated or inactivated before RNA isolation procedures.
12
Question
How can glassware be decontaminated to inactivate RNases?
Answer
Glassware must be decontaminated for 4-6 hours at 400C.
13
Question
How should samples be stored to prevent RNA degradation by intracellular RNases?
Answer
Samples should be kept frozen in liquid nitrogen or immersed in a buffer that inactivates intracellular RNases.
14
Question
What techniques are used to isolate viral RNA from samples like serum or plasma?
Answer
Spin columns or beads are used to isolate RNA in these samples.
15
Question
What technique is commonly used for COVID-19 RNA isolation in laboratories?
Answer
The solid phase technique, such as spin columns or beads, is used for COVID-19 RNA isolation in laboratories.
16
Question
How should the sample be kept for RNA isolation in order to inactivate intracellular RNAses?
Answer
Sample should be kept frozen in liquid nitrogen or immersed in buffer
17
Question
How are bacterial and fungal RNA isolated for RNA isolation?
Answer
Chemical lysis or grinding in liquid nitrogen
18
Question
How can viral RNA be isolated for RNA isolation?
Answer
Directly from serum, plasma, culture medium, or other cell-free fluids
19
Question
What technique is commonly used for COVID-19 RNA isolation in laboratories?
Answer
Solid phase technique
20
Question
From which specimen sources can RNA be isolated and what is the expected yield?
Answer
Blood (1 mL - 35-60 µg), WBCs/mL (1 - 10 µg), Buffy coat (1 mL whole blood - 5-10 µg), Bone marrow (1 mL - 50-200 µg), Cultured cells (10 cells - 50-150 µg), Buccal cells (1 mg - 1-10 µg), Solid tissue (1 mg - 0.5-4 µg), Fixed tissue (1 mm - 0.3-2 µg), Bacterial culture (0.5 mL - 0.7 AU, 10-100 µg)
21
Question
How can RNA be isolated from blood and bone marrow samples?
Answer
Isolate from reticulocytes and separate from WBCs through centrifugation
22
Question
Why should tissues be kept frozen in liquid nitrogen when dissociating them for RNA isolation?
Answer
To prevent degradation of RNA due to its single-stranded nature and the presence of RNAses
23
Question
What tissues, such as pancreas, have many innate RNAses requiring immediate immersion in liquid nitrogen for RNA isolation?
Answer
Tissues with many innate RNAses such as pancreas
24
Question
What is the recommended treatment for a tissue sample from the pancreas to inactivate intracellular RNases?
Answer
It should be immersed immediately in liquid nitrogen.
25
Question
What is the expected yield of RNA from various specimen sources, such as blood, WBCs, bone marrow, cultured cells, buccal cells, solid tissue, fixed tissue, and bacterial culture?
Answer
Blood: 1 mL - 35-100 μg, WBCs/mL: 1-10 μg, Buffy coat: 1 mL whole blood - 5-10 μg, Bone marrow: 1 mL - 50-200 μg, Cultured cells: 10 cells - 50-150 μg, Buccal cells: 1 mg - 1-10 μg, Solid tissue: 1 mg - 0.5-4 μg, Fixed tissue: 1 mm - 3-20 μg, Bacterial culture: 0.5 mL - 0.7 absorbance units, 10-100 μg
26
Question
What are some organic isolation methods for RNA extraction?
Answer
Cell lysis step with detergent or phenol in the presence of high salt (0.2 to 0.5 M NaCl), RNase inhibitors including Guanidine isothiocyanate (GITC) and 2-mercaptoethanol, organic extraction of proteins with acid phenol and chloroform, use of isoamyl alcohol to prevent foaming, addition of DNAse to avoid DNA contamination, maintaining the organic phase at pH 4-6.
27
Question
What are the components of the organic phase in nucleic acid extraction?
Answer
Chloroform (25 parts) and Isoamyl alcohol (1 part)
28
Question
What is the role of Chloroform in nucleic acid extraction?
Answer
Enhance the extraction by denaturation of proteins and promote phase separation
29
Question
What is the ratio of Chloroform to Isoamyl alcohol in the organic phase for nucleic acid extraction?
Answer
25:1
30
Question
Why is DNAse added in the nucleic acid extraction process when only RNA is being extracted?
Answer
To avoid contamination of DNA