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Automation

Quantitative PCR


Quantitative PCR reaction setup is tedious and time consuming. Automation of reaction setup eliminates both human error and contamination associated with manual processing. Sigma-Aldrich offers a full range of automation-compatible, high-throughput Quantitative PCR (QPCR) products for use with either probe-based or SYBR® Green based applications to provide accurate real-time quantification of DNA or RNA templates. These products are conveniently packaged as a 2x ReadyMix™ reagent to include everything necessary for QPCR, leaving only the addition of primers, template, and in the case of probe-specific mixes, fluorescent detection chemistry. Each ReadyMix contains Sigma’s antibody mediated hot start mechanism, JumpStart™ Taq DNA Polymerase, for highly specific amplification.

Sigma’s high-throughput Quantitative PCR offering includes products for:

 



Automation Resources

Automated methods have been developed and validated for Quantitative PCR reaction setup. The following resources are available for download:



Features & Benefits


Rapid Procedure

96 QPCR reactions can be setup in less than 10 minutes.

Enhanced Productivity

Walk-away automation of reaction setup allows you to concentrate on other aspects of your work.

Simplified Handling

Products are packaged as a 2x master mix to include everything necessary for QPCR, leaving only the addition of primers, template, and in the case of probe-specific mixes, fluorescent detection chemistry.

High Specificity

PCR ReadyMix includes JumpStart, an antibody-mediated hot start mechanism for highly specific amplification.




Probe based Quantitative PCR

Ordering Information  
Product Code Product Name Package Size
D6442 JumpStart Taq ReadyMix for High-throughput Quantitative PCR 400 reactions
2000 reactions
D7440 JumpStart Taq ReadyMix for Quantitative PCR 100 reactions
400 reactions
D9191 JumpStart Taq ReadyMix with dUTP for Quantitative PCR 100 reactions
400 reactions
Supporting Reagents  
Dual labeled probes and molecular beacons are available from Sigma Custom Products. Click here for more information.



Performance Characteristics
Amplification of Human Genomic DNA Samples

Figure 1. A PCR master mix containing JumpStart Taq ReadyMix reagent, GAPDH forward and reverse primers, and probe set was added to dilutions of human genomic DNA using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. The dual labeled probe was designed with a FAM reporter and a dark hole quencher. Initial template copy number was 9,000 and diluted 10-fold in subsequent wells. Amplification was carried out for the following concentrations of DNA in 12 replicates: 150 ng/µl, 15 ng/µl, 1.5 ng/µl, 150 pg/µl, and 15 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM® 7700 Sequence Detection Systems.



Cross-Contamination Analysis

Figure 2. To test for cross contamination, a method was developed in which two different master mixes were dispensed across a 96-well plate to alternating wells. The first master mix contained the JumpStart Taq ReadyMix reagent, human genomic DNA (1.5 ng/µl), GAPDH primers, and probe set. The second mix included everything in the first mix except the genomic DNA. All samples were subjected to amplification on the ABI PRISM 7700 Sequence Detection Systems. The numbers shown in above table indicate the values of Cycle Threshold (Ct) in each well. The wells without genomic DNA (in yellow) showed no evidence of amplified PCR products, demonstrating that no cross contamination is present.


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SYBR Green based Quantitative PCR

Ordering Information  
Product Code Product Name Package Size
S4438 SYBR Green JumpStart Taq ReadyMix for Quantitative PCR 100 reactions
500 reactions
S5193 SYBR Green JumpStart Taq ReadyMix without MgCl1 100 reactions
400 reactions
S9194 SYBR Green JumpStart Taq ReadyMix for High-Throughput QPCR 400 reactions
2000 reactions


Performance Characteristics
Amplification of Human Genomic DNA Samples

Figure 3. A PCR master mix containing SYBR Green JumpStart Taq ReadyMix reagent and GAPDH forward and reverse primers was added to dilutions of human genomic DNA template using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. Initial template copy number was 1,200 and diluted 5-fold in subsequent wells. Amplification was carried out for the following concentrations of DNA in 12 replicates: 2 ng/µl, 0.4 ng/µl, 80 pg/µl, 16 pg/µl, and 3.2 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM 7700 Sequence Detection Systems.


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Quantitative RT-PCR

Ordering Information  
Product Code Product Name Package Size
QR0100 SYBR Green Quantitative RT-PCR 100 reactions


Performance Characteristics
 
 
Amplification of Human Total RNA Samples

Figure 4. A PCR master mix containing SYBR Green Taq ReadyMix reagent for Quantitative RT-PCR, GAPDH forward and reverse primers was added to dilutions of human total RNA template using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. Amplification was carried out for the following concentrations of total RNA in 12 replicates: 50 ng/µl, 5 ng/µl, 500 pg/µl, 50 pg/µl, and 5 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM 7700 Sequence Detection Systems.


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