Understanding PCR

A Practical Bench-Top Guide

Sarah Maddocks,Rowena Jenkins

• Verlag: Academic Press
• Jahr: 2016
• ISBN:
  9780128026977
• Thema: Biological Sciences
• Thema: Molecular Biology

Understanding PCR: A Practical Bench-Top Guide gives you all of the information you need to plan your first PCR, from reagents to conditions to analysis and beyond. It is a user friendly book that has step-by-step basic protocols, which can be adapted to your needs. Includes helpful information such as where to order your reagents and basic troubleshooting hints and tips.

• Includes resources for reagents
• Explains basic laboratory preparation
• Provides straightforward experimental protocols
• Incorporates fundamental analytical techniques
• Contains a troubleshooting guide

Chapter 1

Things to Consider Before Preparing to Undertake Your First Polymerase Chain Reaction

Abstract

This chapter provides practical advice on what needs to be addressed before undertaking polymerase chain reaction (PCR). From keeping the workspace nuclease free to recipes and shopping lists; information that is vital know and understand before putting on a lab coat and entering the lab, is described. The reader is guided through the practical aspects of preparing to undertake PCR with examples of reagents provided as well as a list of common manufacturers of PCR reagents. Safety considerations are discussed and thought is given to equipment and reagents that will be required for analysis of the first PCR reaction.

Keywords

Laboratory preparation; Material safety data analysis (MSDS); Nuclease free; Reagents; Safety; Timescales

1.1. Introduction

So, you have decided to tackle the polymerase chain reaction (PCR); this is an extremely powerful technique that will enable you to amplify fragments of DNA. PCR is also incredibly versatile, and once you have mastered the basics, you will be able to apply the techniques you have learned to a host of molecular techniques including genetic profiling, detection, gene expression, and genetic modification. The world of PCR can be overwhelming, from deciding on your primer design, to where to buy your polymerase. It is therefore important that you understand the fundamental requirements necessary to prepare and carry out PCR, to avoid months of frustration or results you can not even begin to comprehend.

There are a number of factors to consider when planning to undertake PCR for the first time, and if these are addressed prior to putting on your lab coat and picking up your pipette, then the whole process will run much more smoothly, meaning you are more likely to be successful. Such considerations include aspects of safety, preparation of reagents, and consideration of reaction parameters and organization of your work area. This chapter will describe some of the considerations you need to address before heading into the lab; it is a good idea to equip yourself with a pen whilst reading through this chapter so that you can begin to plan your first PCR.

1.2. Basic Laboratory Preparation

Saying that it is important to work in a clean, organized, and orderly manner might seem obvious but is imperative for successful PCR. PCR is a powerful procedure in which small quantities of DNA are amplified; the success of PCR can be affected by the presence of contaminants from the environment or due to poor handling of reagents, or PCR inhibitors which are often inherently present as a consequence of DNA preparation. Working in an area that is free from possible sources of contamination or potential inhibitors is therefore paramount. So first, ensure that your bench is free from clutter, including left over experiments, reagents, used gloves, or plastics, to name a few, that could contaminate your reaction. It is usually good practice to wipe over your bench with a standard disinfectant or 70% ethanol before you begin—you could also wipe the barrel of the pipettes you will use. Often people use filter tips for PCR to prevent anything that might be in the barrel of the pipette from being inadvertently introduced into the reaction. Sometimes, people use a dedicated “PCR” set of pipettes, but this is not paramount providing you are confident that yours are clean! You will be handling very small amounts of often colorless liquid, so make sure that your pipettes are calibrated to ensure precision. Most of your PCR reagents will need to be kept on ice, so have an ice bucket to hand and also somewhere to dispose of waste, such as used tips and tubes. It goes without saying that you will need to wear a lab coat and gloves (the latter to avoid contamination of your work by you and to protect you from harmful chemicals)—if you think you have contaminated the gloves you are wearing and that you might transfer this to your PCR, then change your gloves. Below is a photograph showing a bench that is setup for PCR (Fig. 1.1). You will notice the sensible work flow, bringing reagents in from the left, preparing the reaction in the central area of the bench, and disposing of used tips or empty tubes into an appropriate jar which is in easy reaching distance.

To ensure that you remember to add each reagent, it is usually a good idea to write yourself a list (an example list of ingredients with hints and tips are given below; volumes will be addressed later) of the ingredients you will add to your PCR reaction and tick them off as you add them; this is also useful when purchasing reagents so that you do not forget anything.

You will usually be setting up more than one reaction at a time if you are, for example, testing a new set of primers, titrating the template material, or preparing replicate reactions. In this case, it is usually a good idea to prepare a master mix of ingredients in one 1.5 mL tube, i.e., for five reactions multiply the volume of each reagent to add by 5, and then divide this up between the PCR tubes that you have. This avoids inaccuracies during pipetting, or problems if reagents have not properly mixed.

Figure 1.1 A PCR workbench.

When using the PCR recipe list, you will most likely still be planning your PCR “on paper,” designing primers and reaction parameters—if you are using a predesigned set of primers with known reaction parameters you can move onto the wet work and skip to Chapter 3 which describes how to analyze the results of the PCR reaction.

PCR Recipe List, Hints, and Tips

• Template DNA: Decide how you will obtain this; will you use a commercially available kit or an “in-house” extraction? Check the concentration of the template material using either by UV spectroscopy or by electrophoresis with a quantitative molecular weight marker (see Chapter 3).

• Primers (forward and reverse): If you need to design primers, please turn to Chapter 2 for guidance. A good start point for PCR is to begin by adding 100 pmol per reaction, but you can prepare a series of dilutions to find the optimum concentration to work with. Primers usually arrive lyophilized, and the company you purchased them from will tell you how to reconstitute them (see Chapter 2). Once you have reconstituted your primers, make a working stock of 100 pmol/mL, and then you would not be constantly dipping into you “master” stock).

• dNTPs: These are usually purchased individually or as a premixed preparation containing equal quantities of all four bases. These are usually available at stock concentration of 100 mM (but please check the information from the manufacturer before use) which will be diluted upon addition to the reaction.

• MgCl₂/MgSO₄: It is vital for PCR to work; it acts as an enzyme cofactor and also impacts the specificity and stringency of primer annealing. Buffers that are provided with commercially available PCR enzymes will often include Mg²⁺ at an amount that will give a working concentration of 1.5 mM when diluted in the reaction. Some ...

Inhaltsverzeichnis

• Cover image
• Title page
• Table of Contents
• Copyright
• Author Biographies
• Chapter 1. Things to Consider Before Preparing to Undertake Your First Polymerase Chain Reaction
• Chapter 2. Designing and Ordering Your Polymerase Chain Reaction Primers
• Chapter 3. Analyzing Your Template DNA and/or PCR Product
• Chapter 4. Quantitative PCR: Things to Consider
• Chapter 5. Carrying Out Q-PCR
• Chapter 6. Using PCR for Cloning and Protein Expression
• Chapter 7. Polymerase Chain Reaction for Knocking Out Genes
• Appendix 1
• Appendix 2
• Index