CFX Connect Real-Time PCR Detection System from Bio-Rad

March 05, 2013

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University of Virginia
Infectious Diseases
Laboratory and Research Specialist
Standard Curve derived from qPCR amplification of total DNA from mouse stool with Bacteroides primers using the Bio-Rad CFX Connect Real-Time PCR Detection System

Company:

Bio-Rad

Product Name:

CFX Connect Real-Time PCR Detection System

Catalog Number:

185-5201
Image

Our research involves quantitative microbe detection from stools shed by mice – either pathogen or intestinal flora. In order to determine the amount of microbe shed we require a robust and efficient system where bacterial DNA can be quantified. The device works by measuring florescence of non-specific dyes which intercalate into double-stranded DNA after every PCR cycle. This allows one to compare the number of cycles it takes to reach the detection threshold of the machine for your test sample to either a known standard (like 10^8 E coli bacteria) or an endogenous control (such as a housekeeping gene like beta actin).

Experimental Design and Results Summary

Application

qPCR for pathogen detection

Starting Material

DNA extracted from mouse stools

Protocol Overview

In our lab, we have compared the effects of a low-protein diet on Clostridium difficile infection when compared with a standard, normal protein diet. This is accomplished by measuring the amount of Clostridium difficile shed in 10 µg of stool (usually 10^8 or less). We homogenize mouse stools which are then extracted for total DNA content. We load 10 µL of Kapa qPCR master mix with SYBR green (Kapa Biosystems, catalog number KK4602), 4 µL of our sample, 4 µL of nuclease-free water, and 1µL of each of our forward and reverse primers into a 96-well qPCR plate along with a dilution series of known standards (usually DNA isolated from 10^2 through 10^8 bacteria). All samples are run in duplicate. The plate is then sealed and inserted into the Bio-Rad CFX Connect machine.

Tips

None

Results Summary

Our results have been reproducible within duplicates of samples – we rarely have duplicates that vary by more than 0.5 cycles.

Features Summary

The Bio-Rad CFX Connect Real-Time PCR Detection System is a standard, 96-well quantitative PCR detection system that can detect up to two fluorophores at the same time. Detection thresholds for quantifying microbes run from 10^3 to 10^8. CFX Manager is the software which allows you to operate the CFX connect, and runs from a separate computer which is hooked up to the CFX Connect. The software to run the machine is very easy to use and intuitive. You choose time and temperature for your initialization, denaturation, annealing, and hold steps, as well as the cycle number. You then go to the well map and choose your samples and standards, as well as your flourophores. Then hit run and you’re done! Depending on the program you use, you can have results in less than 90 minutes. If you are comparing your test samples to a known standard, a standard curve will be generated as well as quantification results to your test samples. All of this data can be exported to a Microsoft Excel spreadsheet with a simple click. You will also be given an efficiency rating for your standard curve which will allow you to determine if your standard curve worked correctly (an efficiency rating of 80-120% is optimal).

Additional Notes

The opening and closing of the lid of the machine is automated. Use the computer controls (located within the CFX Manger program) to open and close the machine rather than forcing it open to load your sample.

Image Gallery

Standard Curve derived from qPCR amplification of total DNA from mouse stool with Bacteroides primers using the Bio-Rad CFX Connect Real-Time PCR Detection System

Summary

The Good

Intuitive, easy to use, compact, and affordable.

The Bad

Lid must be closed automatically even though it can be forced open manually – the lid is of similar design to regular PCR machines which are opened manually so it can be confusing.

The Bottom Line

This is a great little machine for doing your routine qPCR work. Bio-Rad also has great support for their products so if you do run into a problem, they will be happy to help!

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