BIOS.113.Lab.03.DNA.isolation.S25

Lab Overview

• Course: BIOS 113, Spring 2025

• Lab Title: Isolation of Genomic DNA from Cells

• Safety Precautions:

  • Wear pants and closed-toed shoes.

  • Avoid contact lenses during lab.

  • Use gloves and safety glasses when handling chemicals, especially phenol.

Background on DNA Isolation

• Objective: To isolate genomic DNA (gDNA) from various cells, particularly peas.

• Cell Lysis:

  • Cells are lysed to expose cellular contents and liberate DNA.

  • SDS (sodium dodecyl sulfate) is used for cell membrane disruption and protein denaturation.

• Nucleic Acid Purification:

  • After lysis, RNA removal occurs to isolate pure gDNA.

  • Process involves using phenol/chloroform extraction to separate nucleic acids from proteins and lipids.

Steps in DNA Isolation

A. Lysing the Cell: Making Crude Pea Lysate

1. Materials Needed:

   • Blender, 100mL TE buffer (kept on ice), cheese cloth.

   • 500mL beaker, 1.5mL centrifuge tubes, 10% SDS.

   • Protease powder, 100mL Pisum sativum (pea) samples.

2. Procedure:

   • Blend 100mL of TE buffer with 100mL of peas for 1 minute to break cell membranes.

   • Strain through cheesecloth into a 500mL beaker.

   • Transfer 620μL of the crude lysate to two 1.5mL centrifuge tubes.

   • Add 70μL of 10% SDS and 10μL of protease solution to each tube.

   • Gently rock the solution every minute for 10 minutes to ensure thorough mixing.

B. Isolating and Extracting the DNA

1. Preparation:

   • Gather phenol:CHCl3, centrifuge, sterile gloves, and safety glasses.

   • Ensure proper safety precautions are observed, work under supervision if necessary.

2. Procedure:

   • Add equal volume (~700μL) of phenol:CHCl3 to each lysate solution to separate layers.

   • Gently rock for 2 minutes, then centrifuge for 2 minutes at 13,000 rpm and 4°C.

   • Identify the two layers formed: organic layer (bottom) with proteins/lipids and aqueous layer (top) containing DNA.

C. Concentrating the Nucleic Acids

1. Required Materials:

   • 3M Sodium Acetate, 95% EtOH, 75% EtOH, nuclease-free water.

2. Steps:

   • Add 50μL of 3M Sodium Acetate and 900μL of 95% EtOH to the aqueous solutions obtained from the previous step and mix gently.

   • Centrifuge at 13,000 rpm for 10 minutes; expect small white or clear DNA/RNA pellets.

   • Remove the supernatant and add 75% EtOH without mixing.

   • Centrifuge again for 5 minutes, discard supernatant, and allow pellets to dry.

   • Re-dissolve DNA/RNA pellet in 100μL nuclease-free water.

D. Quantifying DNA/RNA Purity

1. Materials Required:

   • Cuvette, transfer pipette, UV spectrophotometer.

2. Procedure:

   • Zero the spectrophotometer with a blank cuvette filled with nuclease-free water.

   • Dilute the DNA solution 1:40 (10μL DNA solution + 390μL water).

   • The instructor will pipette diluted solution into a cuvette and take absorbance readings at OD260 and OD280.

3. Analysis:

   • Calculate the ratio of OD260 / OD280:

     • A ratio close to 1.8 indicates high nucleic acid purity.

     • A lower ratio suggests protein or contaminant presence; repeat previous processes if needed.