LSI Quantitative PCR Core (QPCR Core)
Equipment available
The use of equipment and training sessions are free of charge.
BioRad Opticon 2
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_07218.pdf
QuantStudio 3
QX200 Droplet Digital PCR System with AutoDG 1
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf
Bio-Rad has developed a unique PCR technology that quantifies DNA molecules by Droplet Digital PCR. Droplet Digital PCR provides an absolute measure of target molecules with unrivaled accuracy, precision, and sensitivity. Applications include copy number variation, mutation detection, absolute quantitation, rare sequence detection, gene expression analysis, and much more.
Compared to conventional real-time PCR systems, Droplet Digital PCR offers:
– Unparalleled precision, 10x greater precision than real-time PCR, (+/- 10%)
– Detection of rare targets in complex backgrounds, 100x more sensitive than real-time PCR
– Absolute and Direct concentration measurement reported in Copies/ul
– Standard curves are not required with Droplet Digital PCR and no more Ct or ΔΔCT values.
To overcome the limitations associated with Real-time PCR, droplet digital PCR (ddPCR) was introduced. The technique is based on the partitioning of a single sample (containing DNA, primers, polymerase, and a fluorescent probe or dsDNA binding dye chemistry) into many thousands of PCR reaction vessels. These thousands of PCR reactions are amplified in a standard 96 well thermal cycler.. Each reactor can be then be sorted by their fluorescent signal as positive or negative delineating the presence or absence of target. This simple readout of reactor partitions as a binary code of ones (positive) and zeroes (negative) and therefore the readout is digital. Given that the presence of target in a given reactor is a random event, the associated data fits to a Poisson distribution. This permits the determination of absolute DNA copy numbers in a sample without the requirement of generating a standard curve and since the data are acquired at the end-point, the results are minimally affected by changes in reaction efficiency. The QX200 Droplet Digital PCR system uses emulsion chemistry to partition 20uL samples into 20,000 partitions. This high level of partitioning serves to provide a wide dynamic range of quantitation as well as provides high level of precision and sensitivity, while eliminating many of the requirements for optimization and validation typically associated with Real-time PCR. The Bio-Rad QX200 instrument is the most cost-effective, highest throughput, digital PCR system on the market.
Manager Aaron Bogutz Blair aaron.bogutz@ubc.ca
Location LSI 5th floor, East Wing