Real-Time PCR


The Genomics Core Facility (GCF) has four Applied Biosystems 7900 Sequence Detection System instruments. The instruments may be used for gene expression, allelic discrimination (SNP analysis), and quantitative pathogen detection. The instruments are equipped to run 96 well and 384 well plates and TaqMan Low Density Arrays (TLDA).

For 384 well plate experiments, the Perkin Elmer Multiprobe II EX pipetting robot is programmed to custom pipet quantitative PCR plates. Please see Robotics for further information.

There is a $10 per run charge for use of an instrument. GCF personnel provide both informal and formal training, provide troubleshooting advice, and maintain and calibrate the instruments at no charge to the user.

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Users of the instruments schedule an appointment for run time on the instrument, set up their analysis plates, and run them at their reserved time.

The maximum appointment time available for a single run is three hours. Please schedule a separate appointment for each real-time PCR run. If you are more than thirty minutes late beginning your run, please store your plate refrigerated and protected from light and reschedule your run. Both RNA and cDNA plates may be safely stored in this manner up to 48 hours. Please do not inconvenience others by running past your appointment time.

Appointments may be reserved by Pennington employees using the Outlook calendar. The instrument resources are listed in Outlook as R_GCF_Aphrodite, R_GCF_Hera, R_GCF_Castor, R_GCF_Pollux. The current plate configuration for each instrument may be found at the top of the Outlook calendar.

Detailed instructions for using the Outlook calendar to schedule an instrument (resource) may be found in the Scheduling Resources - Frequently Asked Questions.

Persons outside of Pennington may request a reservation by contacting GCF.

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A single real-time PCR run is $10.00.

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Training courses are conducted periodically. They are announced through this web site and via email. Individual consultation for experimental design, primer design, troubleshooting or training may be scheduled by contacting GCF.

Applied Biosystems offers live, online Web-based Training Series for Real-Time PCR. To see when the next series is offered, please check the Training website and search for "Real-Time Quantitative PCR Training Series".

For PBRC employees, documents from the summer series of courses can be found on the shared drive O:\GCF\User Information\Real-Time QPCR\Real-Time PCR Course_AB-WEBINAR

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SDS and Primer Express Software

Two workstations in room L2012 have SDS software for analysis of plates run on the 7900 and Primer Express software for quantitative PCR primer design. These workstations are freely available on a first-come, first-served basis. It is not necessary to schedule an appointment for workstation use.

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DataAssist Software

DataAssist is a data analysis tool for sample comparison when using the comparative CT (ΔΔCT) method for calculating relative quantitation of gene expression. It contains a filtering procedure for outlier removal, various normalization methods based on single or multiple genes, and provides relative quantification analysis of gene expression through a combination of statistical analysis and interactive visualization.

Click here to download.

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