cell bio lab 6

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Questions and Answers

What is the absorbance maximum for nucleic acids?

280 nm

260 nm (correct)

350 nm

230 nm

What A260/A280 ratio indicates pure DNA?

0.8–1.0

1.8–2.0 (correct)

2.1–2.3

1.5–1.7

What might a lower A260/A280 ratio indicate?

Contamination with residual phenol (correct)

High DNA purity

Excess DNA concentration

Complete RNA extraction

Which of the following is NOT a precaution for working with RNA?

Use regular pipette tips and tubes Signup and view all the answers

What is the significance of maintaining RNA samples on ice during use?

To prevent RNase degradation Signup and view all the answers

How should RNA samples be stored for long-term preservation?

At –80°C Signup and view all the answers

Which of the following actions helps control the source of RNase?

Using DEPC water for inactivation Signup and view all the answers

What should be included in the lab report's results section?

The concentration of extracted genomic DNA Signup and view all the answers

What is the primary purpose of adding isopropanol during DNA extraction?

To precipitate the DNA Signup and view all the answers

Which component is crucial for preventing DNA degradation during extraction?

EDTA Signup and view all the answers

What role does potassium acetate play in the DNA extraction process?

It neutralizes the lysis solution's high pH. Signup and view all the answers

Which of the following is NOT an ingredient in the extraction buffer?

Lysate Signup and view all the answers

What function does 70% ethanol serve in the DNA extraction process?

It washes the DNA. Signup and view all the answers

How is the purity and concentration of genomic DNA extracts typically measured?

NanoDrop spectrophotometer Signup and view all the answers

What is the role of β-mercaptoethanol in the extraction buffer?

To reduce disulfide bonds in proteins. Signup and view all the answers

Which of the following accurately describes the function of NaCl in the extraction buffer?

To stabilize the DNA. Signup and view all the answers

purpose of tris-hcl

maintain the pH at the optimal condition for DNA extraction Signup and view all the answers

purpose of EDTA

EDTA binds to metal ions, like magnesium and calcium, which are cofactors of DNase; thus preventing the degradation of DNA Signup and view all the answers

purpose of NaCl

dissolves the DNA and maintains its stability Signup and view all the answers

purpose of 1% SDS

SDS causes protein denaturation and disrupts the interactions between proteins and lipids in cell membranes, causing the cell to break down and release its contents Signup and view all the answers

Potassium acetate (Bio 101 Solution III) PURPOSE

Creates a high-salt environment, and neutralizes the high pH of the lysis solution, facilitating the precipitation of SDS-bound proteins and other cellular debris. Signup and view all the answers

Isopropanol / ethanol PURPOSE

The isopropanol facilitates the interaction of K+ or Na+ with the PO3− groups on the sugar phosphate backbone of nucleic acids. This interaction makes the DNA molecule less soluble in water, precipitating the DNA. Signup and view all the answers

working with rna...

Keep the RNA samples intact by placing on ice while in use <ul> <li>For long term storage: store RNA samples in –80°C Control the source of RNase</li> <li>Use RNase-free pipette tips and microcentrifuge tubes Clean work area and pipettes with RNaseAway Wear clean gloves, masks and lab coat Inactivate the RNase using DEPC water</li> </ul> Signup and view all the answers

order of operations for rna extraction in plants

lysing, filtering, binding, washing, eluting, RNA!!! Signup and view all the answers

Study Notes

Lab 6: Genomic DNA and Total RNA Extraction (Demo)

Lab Objectives (Genomic DNA):
- Understand genomic DNA extraction principles from plant tissue.
- Extract genomic DNA from Arabidopsis thaliana leaf samples.
- Measure and assess the purity of extracted genomic DNA.
Lab Objectives (Total RNA):
- Learn precautions for working with RNA.
- Understand RNA extraction principles.

DNA Extraction Principles (Plants)

Tissue Preparation: Grind tissue in lysis buffer to disrupt cell walls and membranes.
Separation: Centrifuge to separate DNA from the organic phase (containing lipids and proteins).
Precipitate & Wash: Add isopropanol to precipitate DNA, then wash with ethanol to remove impurities.
Solubilize: Add water to solubilize the DNA.

Extraction Buffer Components

Tris-HCl (1.50 mM): Maintains optimal pH for DNA extraction.
EDTA (2.10 mM): Binds metal ions (Mg²⁺, Ca²⁺), preventing DNase degradation of DNA.
NaCl (3.100 mM): Dissolves DNA and maintains stability.
SDS (1.0%): Denatures proteins, disrupts cell membranes, releasing DNA.
β-mercaptoethanol (5.10 mM): Strong reducing agent that breaks disulfide bonds in proteins, removing protein contamination.

Potassium Acetate and Isopropanol/Ethanol

Potassium Acetate: Creates a high-salt environment to neutralize lysis solution pH, facilitating precipitation of SDS-bound proteins and cellular debris.
Isopropanol/Ethanol: Facilitates interaction of K⁺ or Na⁺ ions with phosphate groups on DNA, reducing DNA solubility in water and causing precipitation.

DNA Quantification & Qualification

NanoDrop Spectrophotometer: Measures DNA concentration and purity.
A260/A280 Ratio: Indicates DNA purity:
- Pure DNA: Ratio of 1.8-2.0
- Lower ratios indicate contamination (residual phenol, sugar, or reagents).
- Nucleic acids absorb at 260nm, proteins at 280nm.

RNA Precautions

Storage: Store RNA samples at -80°C for long-term preservation. During use keep RNA samples on ice to maintain integrity.
RNase Control: Control RNase sources, use RNase-free pipettes, tubes and clean all work surfaces.
Safety: Wear gloves, masks and lab coats for protection.
DEPC Water: Use DEPC water to inactivate RNase.

RNA Extraction Principle (Plants)

Lysing: Extract cellular RNA.
Filtering: Remove cell debris.
Binding: Bind RNA to a specific medium.
Washing: Remove impurities.
Eluting: Release the purified RNA using RNase-free water.

RNA Assessment

Assessment methods: Visual inspection using gel electrophoresis, quantification with spectrophotometry methods to assess quality.

Lab 6 Report Requirements

Introduction: Explain the principle and purpose of genomic DNA extraction.
Materials: List all chemicals and instruments, including concentrations.
Methods: Describe the experimental steps performed.
Results: Report extracted genomic DNA concentration, A260/A280 ratio, and unexpected findings.
Discussion/Conclusion: Interpret results and discuss potential improvements; give significance of the lab work.

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Description

This quiz covers the objectives and principles of extracting genomic DNA and total RNA from plant tissues, particularly focusing on Arabidopsis thaliana. Understand the detailed steps in the extraction process, precautions needed when handling RNA, and the roles of various buffer components.