JoVE Lab Manual
Biologia
DNA Isolation and Restriction Enzyme Analysis

DNA Isolation and Restriction Enzyme Analysis

CONCEITOS
PREPARAÇÃO DO INSTRUTOR
PROTOCOLO DO ALUNO
Lab Manual
Biologia
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Lab Manual Biologia
DNA Isolation and Restriction Enzyme Analysis

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02:15 min

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January 29, 2019

Learning Objectives

What are the essential steps of DNA isolation?

The essential steps of DNA isolation are cell harvesting, lysis, protein degradation, and DNA precipitation.

How is the quantity and purity of DNA determined?

Scientists use a spectrophotometer, which measures the intensity of a light beam passed through a sample as a function of its wavelength. By quantifying the output of wavelengths that are specific to nucleic acids or impurities, one can not only quantify the amount of DNA but also estimate the purity of the DNA sample.

What is a restriction enzyme?

It is an enzyme of bacterial origin that can recognize specific DNA sequences and cut it into segments at those locations.

How is gel electrophoresis used for DNA analysis?

Scientists can load DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size.

Why is genetic profiling important?

DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics. In addition, these advancements recently paved the way for personalized medicine with improved treatment outcomes.

List of Materials

Print Materials List
  • Popsicle sticks (1/group)
    5
  • Glass funnels (1/group)
    5
  • 15 mL conical tubes (2/group)
    10
  • Drinking cups (1/group)
    5
  • 1 % NaCl solution (10 mL in a drinking cup/group)
    50
  • Permanent marker (1/group)
    5
  • Beaker for collecting waste (1/group)
    5
  • Lysis buffer I: TE buffer with SDS (TES Buffer) (1 mL/group)
    5 mL
  • Lysis buffer II: TE buffer without SDS (1 mL/group)
    5 mL
  • Proteinase K (10 µL/ group)
    50 µL
  • 2.5 M NaCl (100 µL/group)
    500 µL
  • Ethanol 100 % (4°C) (1 mL/group)
    5 mL
  • Glass rods (2/group)
    10
  • Stopwatch (1/group)
    10
  • Microfuge tubes (1.5 mL) (a beaker full/station)
    1 per station
  • Floating foam microfuge racks (1/station)
    1 per station
  • Cell phone with a camera
    1 per group
  • Automated pippete sets (1 µL-1 mL)
    -1 Dependent on the lab size
  • Pipette tips (1 µL-1 mL) 1box each/group
    -1 Dependent on the lab size
  • Microfuge centrifuge
    -1 Dependent on the lab size
  • Water bath (set to 56°C)
    -1 Dependent on the lab size
  • Nuclease free water (125 µL aliquot/group)
    125 µL
  • Lamda DNA (12 µL/group)
    12 µL
  • NEB buffer 2.1 (15 µL/group)
    15 µL
  • Restriction enzyme 1 (StuI) (1 µL/group)
    1 µL
  • Restriction enzyme 2 (BsrGI) (1 µL/group)
    1 µL
  • Stop watch (1/group)
    5
  • Enzyme cooler
    -1 Dependent on the lab size
  • Water bath (set to 37°C)
    -1 Dependent on the lab size
  • Computer with internet connection
    -1 Dependent on the lab size
  • Agarose Powder
    -1 Dependent on the lab size
  • Tris-Acetate-EDTA buffer
    -1 Dependent on the lab size
  • Syber safe gel stain (10,000X) (10 µl/100mL gel)
    -1 Dependent on the lab size
  • 6X loading dye
    -1 Dependent on the lab size
  • 1 kb DNA Ladder
    -1 Dependent on the lab size
  • Weighing balance
    1 whole class
  • Microwave
    1 whole class
  • DNA gel apparatus
    1 whole class
  • UV transilluminator
    1 whole class

Preparação do Laboratório

Print Setup Guide
  1. Preparation of Solutions and Materials
    • NOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
    • First gather the necessary materials, including popsicle sticks, funnels, 15 mL conical tubes, micropipettes, ice buckets with ice, small cups and microcentrifuge tubes.
    • Next set the water bath used in part one of the laboratory exercise, to 56 °C.
    • Set a second water bath to be used in part two of the laboratory exercise to 37 °C.
    • To make TE buffer, first measure out 400 mL of RO water.
    • Then add five mL of 1M Tris-HCL pH 8, and then 1 mL of 0.5 M EDTA solution.
    • Next, add sufficient RO water to bring the solution up to 500 mL.
    • To make TE buffer with SDS or TES buffer, again measure out 400 mL of RO water.
    • Add five milliliters of 1M Tris-HCL pH 8, and then add 1 mL of 0.5 molar EDTA solution.
    • Now add 5 mL of 10% SDS solution.
    • Bring the volume up to 500 mL, using RO water.
    • Next, prepare the 1% saline solution that the students will use to collect cheek cells. In an empty, clean soda bottle, dissolve 5 g of NaCl in 500 mL of RO water.
    • Pour 10 mL aliquots into small drinking cups for the students to use, one per student group.
    • After this, prepare a sufficient quantity of 10 mL aliquots of 100% ethanol for the students to use. NOTE: Store these ethanol aliquots in the freezer or refrigerator to keep them preferably ice-cold.

Tags

JoVE LabLab: 12 Prep