Part a

Basic Techniques & Equipment

This is Part A, Basic Techniques and Equipment, under the module topic Molecular Biology Laboratory Equipment. This topic part has three sections to review: Content Tutorial, Animations, and Activities.

Content Tutorial

Introduction

Please review the following Laboratory Tour web link provided by the Massachussetts Institute of Technology Open Courseware project. This resource will provide you with a basic overview of the equipment that you will encounter in most molecular biology laboratories.

• MIT “Lab Tour” (ocw.mit.edu, Multimedia Page)

Basic Techniques & Equipment

a. Micropipettes
Pipettes are used to accurately measure and dispense small volumes of liquid. The capacity of a micropipette can range from less than 1µl to 1000µl (1ml), while macropipettes can measure volumes greater than 1ml. These are used to physically interact with microscopic samples, such as in the procedures of microinjection and patch clamping. Most micropipettes are made of borosilicate, aluminosilicate or quartz with many types and sizes of glass tubing being available. Each of these compositions has unique properties which will determine suitable applications.
Micropipettes brands include: Eppendorf, Hamilton, Rainin, Drummond, BrandTech, Oxford, Hirschmann, Biohit, Labnet, Nichiryo, Gilson, Corning, VistaLab, Thermo, Jencons, Vertex, Handypett, Pricisexx

PDF Document

• Lab Techniques: “Using a Micropipettor (www.umsl.edu, PDF Document)

b. Spectrophotometry
In physics, spectrophotometry is the quantifiable study of electromagnetic spectra. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared. Also, the term does not cover time-resolved spectroscopic techniques. Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color (or more specifically the wavelength) of light. Important features of spectrophotometers are spectral bandwidth and linear range of absorption measurement. The most common application of spectrophotometers is the measurement of light absorption.

Web Resource

• Principles of Spectrophotometry (www.ruf.rice.edu, Multimedia Page) Reference: David R. Caprette, Rice University *Note: when viewing the webpage, do not go on to the “next” section, you will re-visit this site later in the Protein Techniques Module.

c. Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Molecular Biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid”. The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.
The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or g. The particles’ settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity.

Web Resources

• Differential Centrifugation (www.ruf.rice.edu, Multimedia Page)
• Centrifuge Safety Guidelines (www.hawaii.edu, PDF Document) Reference: University of Hawaii, John A. Burns School of Medicine EHSO

d. Autoclave
An autoclave is a device to sterilize equipment and supplies by subjecting them to high pressure steam at 121° C or more. There are two main types of Autoclaves:
Stove top autoclaves actually resemble a pressure cooker. Each unit comes complete with a bolt-down lid and a pressure gauge on the outside. These units require an outside heat source and can be extremely dangerous in untrained hands. These should only be used by trained laboratory professionals
Front loading autoclaves are more widely used for their convenience, but must also be handled with great care. The units are box-shaped and self contained, equipped with a heating unit to turn water into vapor for sterilization. The autoclave’s controls allow the operator to set the desired temperature, and determine how long the machine will remain in operation. There is also a gauge to track chamber temperature/pressure.

Web Resource
Standardized Autoclave Protocol(www.hawaii.edu, PDF Document) Reference: University of Hawaii, John A. Burns School of Medicine

e. Microscopes – dissecting, inverted, fluorescent, confocal
A microscope (also known as compound microscope) is an instrument to see objects too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope.
Dissecting microscopes, also knows as stereo or stereoscopic are low power magnification for dissection.
An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope.
A fluorescent microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.
Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.

Web Resources

• Light Microscopy Comparison of Optics, by David R. Caprette, Ph.D. (www.bioedonline.org, PowerPoint Document)
• Measuring and Counting with a Light Microscope, by David R. Caprette, Ph.D.(www.bioedonline.org, PowerPoint Document) *Note, the last 4 slides of this presentation are a useful reference for Module IV. Cell Culture Techniques for cell counting

Animations

The Microscope

• Microscope Animation #1 (nobelprize.org, Multimedia Document)
• Microscope Animation #2 (nobelprize.org, Multimedia Document)

Activities

1. Basic Skills– In the following resource link you will performing activities involving the concepts and calculations for dilutions and solutions, the use and calibration of micropipettesm, and doing a spectrophotometric analysis of food colors.
Basic Skills: Dilutions, Micropipetes and Spectrophotometry (bio.classes.ucsc.edu, PDF Document)

2. Micropipette Technique– In the following resource link you will be performing a pipette simulation where you will be working with Polymerase Chain Reaction. This simulation provides a list of the components that you will need to perform the PCR in addition to components that you will need to micropipette your solutions.
Virtual Technique Practice: Micropipette Technique (udel.edu/present/Becky/lehman, Multimedia Page)

3. Lab Equipment Uses– Click on the link below to visit an activity on laboratory equipment utilization. This activity will help familiarize you the various equipment that you may encounter in a molecular biology laboratory.
Lab Equipment Uses (sciencewithsandy.com, Multimedia Page)