Richard Lab:qPCR

Introduction

Quantitative PCR (qPCR or also called Real-time PCR) is a very useful protocol for determining comparitive populations in a sample. This protocol is primarily for comparing bacterial populations in environmental samples (like silage), but can be used for other purposes as well. RT-qPCR can also be used to quantify and compare RNA in a sample including comparing relative mRNA levels. For more information on qPCR and its uses, please see the qPCR hub page or the PCR hub page.

DNA Preparation

• Your DNA can be extracted from any sample. We commonly use a kit for this purpose, but there are a variety of methods around to extract DNA from many materials.
• An important consideration is that the DNA is free of humic acids, which are common contaminants of dna extracted from soil or silage samples. Humic acids will screw up your qPCR.
• Typically 1-20ng of sample need to be added to each reaction. This will generally involve diluting your DNA.

PCR Mix

While there are many commercially premixed 2X PCR solutions to choose from, a cheap and easy homemade stock is really useful for most applications.

2X SYBR Mix

This recipe is to make 1ml of 2X qPCR master mix using Taq DNA polymerase (with thermopol buffer) available from New England Biolabs. This mix is enough to make an entire 96 well plate of 20μL qPCR reactions.

• 730μL Water
• 200μL Thermopol Buffer (with Mg²⁺)
• 50μL dNTPs
• 10μL Taq DNA Polymerase (~15 units)
• 7μL each primer (100μM)
• 3μL SYBR Green (100x)