Reliable Real-Time PCR Cycler

May 11, 2018

The Scripps Research Institute
The Cell and Molecular Biology
Assistant Professor

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Company:

Bio-Rad

Product Name:

CFX Connect™ Real-Time PCR Detection System

Catalog Number:

185-5201

Real-time PCR systems are required to quantitatively determine the levels of gene expression in the cells or tissues. We use PCR cyclers also to determine the levels of human-specific Alu repeats within the DNA purified from animals bearing human tumor xenografts. In combination with a standard curve, this approach allow us to determine the levels of tumor cell dissemination in cancer metastasis models. The CFX Connect Real-Time PCR Systems (#185-5201 or 185-5200) are designed to detect 1 target or 2 targets simultaneously per well of a 96-well plate. We purchased the CFX Connect Real-Time PCR Detection System with a starter package (#185-5200), containing SYBR Green Supermix, 96-well PCR plates and plate seals, which is a highly recommended option.

Experimental Design and Results Summary

Application

Real-time human-specific Alu PCR.

Starting Material

Genomic DNA purified from the lungs or other tissues harvested from animals bearing human tumor xenografts.

Protocol Overview

We use 10 ng DNA in 3 microliters and 7 microliters of "master mixture" per well of a PCR plate. Master mixture contains 2 primers (forward and reverse), each at 0.4 microliters, 2.5 microliters of SYBR green from Roche and 3.7 microliters of DEPC water. Our PCR protocol includes 1 step: 95oC for 3 min, and 40 X 30-sec cycles of 95oC-63oC-72oC.

Tips

We use 50% less CYBR green than recommended by the manufacturer with no change in the sensitivity of detection.

Results Summary

At the end of the assay, the PCR cycler creates a report protocol indicating all parameters that you chose from available options. Basically, you get the table indicating the number of cycles (Cq) that were required to detect fluorescence in each well: the lower Cq values mean higher initial copy numbers of the target. By using a standard curve created by running the DNA purified from the samples containing increasing numbers of human tumor cells spiked into constant number of host cells (e.g. murine embryo or chick embryo fibroblasts), we are able to quantitatively determine actual numbers of human tumor cells metastasized from a primary tumor into secondary tissues of a tumor-bearing animal (e.g. lungs of a mouse).

Features Summary

The CFX Connect Real-Time PCR Detection System is available with a starter package containing SYBR Green Supermix, 96-well PCR plates, and plate seals (185-5200), which is a highly recommended option.

DOI or PMID #

N/A

Additional Notes

The image shows the CFX Connect PCR machine and a PCR plate, allowing to appreciate the compact size of the system.

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Summary

The Good

The Bio-Rad CFX-Connect PCR machine is a compact, affordable and a highly reliable system for every day PCR running. Bio-Rad also provides excellent technical support if needed.

The Bad

None detected, but the machine should be on for some time before the PCR program is open.

The Bottom Line

We run PCR reactions almost every day in our lab and this Bio-Rad machine proved to be reliable system generating highly reproducible results.

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