Reaction size guidelines for qPCR

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Real-time PCR

Product Overview

Real-time PCR (qPCR) is a powerful technique for analyzing samples to quantify target sequences or gene expression. qPCR is typically performed using reactions containing the sample template, master mix, forward and reverse primers, and often ROX reference dye, diluted in PCR-grade water. The volume of each reaction is dependent on experimental conditions, particularly the size of the reservoir in the plate or system being used. For instance, 96-well plates typically utilize 20-μl reactions, 384-well plates typically utilize 10-μl reactions, 1,536-well plates typically utilize 2-μl reactions, and high-throughput systems, like the SmartChip Real-Time PCR System, utilize volumes as low as 100 nl.

Typically, kits and master mixes report a product size based on number of reactions. However, as discussed above, reaction volumes can vary, resulting in differing numbers of reactions from a given kit based on the reaction volume utilized. This can be further complicated depending on the concentration of the starting master mix (e.g., 2X, 5X, etc.). To help clarify exactly how many reactions can be obtained from a given kit, we have provided the tables below (separated based on the general product type). Each lists the specific catalog number, total volume and concentration of master mix included, and number of reactions obtained with frequently utilized reaction volumes.

One-step RT-qPCR kits Two-step RT-qPCR kits Probe-based qPCR kits TB Green dye-based qPCR kits

One-step RT-qPCR kits

One-step reverse transcription kits perform the reverse transcriptase step in the same tube as the qPCR reaction. We offer kits for both probe- and TB Green-based detection. All master mixes included are in a 2X format.

One-Step PrimeScript RT-PCR Kit (Perfect Real Time): number of reactions based on reaction volume

Catalog #	2X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
RR064A	3 x 840 µl (2.52 ml)	100	200	250	500	1,000	2,500	5,000
RR064B	15 x 840 µl (12.6 ml)	500	1,000	1,250	2,500	5,000	12,500	25,000

One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time): number of reactions based on reaction volume

Catalog #	2X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
RR086A	3 x 840 µl (2.52 ml)	100	200	250	500	1,000	2,500	5,000
RR086B	15 x 840 µl (12.6 ml)	500	1,000	1,250	2,500	5,000	12,500	25,000

Two-step RT-qPCR kits

Two-step reverse transcription kits perform the reverse transcriptase step to generate cDNA before adding the cDNA to the PCR, and can be combined with our probe- and dye-based qPCR kits for full analysis. All master mixes are in a 5X format.

PrimeScript RT Master Mix (Perfect Real Time), PrimeScript RT Reagent Kit (Perfect Real Time), and PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time): number of reactions based on reaction volume

Catalog #	5X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
RR036A	400 µl	40	80	100	200	400	1,000	2,000
RR036B	4 x 400 µl (1.6 ml)	160	320	400	800	1,600	4,000	8,000
RR037A	400 µl	40	80	100	200	400	1,000	2,000
RR037B	4 x 400 µl (1.6 ml)	160	320	400	800	1,600	4,000	8,000
RR047A	400 µl	40	80	100	200	400	1,000	2,000
RR047B	4 x 400 µl (1.6 ml)	160	320	400	800	1,600	4,000	8,000

Probe-based qPCR kits

Our probe-based qPCR kits give high specificity, sensitivity, and reproducibility. All master mixes are in a 2X format.

Premix Ex Taq DNA Polymerase (Perfect Real Time), Premix Ex Taq (Probe qPCR), Premix Ex Taq (Probe qPCR), ROX Plus: number of reactions based on reaction volume

Catalog #	2X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
RR039A	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR039B	2 x 5 ml (10 ml)	400	800	1,000	2,000	4,000	10,000	20,000
RR039W	25 ml	1,000	2,000	2,500	5,000	10,000	25,000	50,000
RR390A	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR390B	2 x 5 ml (10 ml)	400	800	1,000	2,000	4,000	10,000	20,000
RR390L	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR390W	5 x 5 ml (25 ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000
RR39LR	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR39WR	5 x 5 ml (25 ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000

TB Green dye-based qPCR kits

Our dye-based qPCR kits utilize our proprietary TB Green intercalating dye. This versatile dye can be used with normal methods and equipment with no need for protocol modifications. All master mixes are in a 2X format.

TB Green Advantage qPCR Premix and Terra qPCR Direct TB Green Premix: number of reactions based on reaction volume

Catalog #	2X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
639676	4 x 625 µl (2.5 ml)	100	200	250	500	1,000	2,500	5,000
638319	5 x 1 ml (5 ml)	200	400	500	1,000	2,000	5,000	10,000

TB Green Premix Ex Taq (Tli RNase H Plus) and TB Green Premix Ex Taq II (Tli RNase H Plus): number of reactions based on reaction volume

Catalog #	2X master mix volume	50-µl rxns	25-µl rxns	20-µl rxns	10-µl rxns	5-µl rxns	2-µl rxns	1-µl rxns
RR420A	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR420B	2 x 5 ml (10 ml)	400	800	1,000	2,000	4,000	10,000	20,000
RR420L	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR420W	5 x 5 ml (25 ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000
RR42LR	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR42WR	5 x 5 ml (25ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000
RR820A	5-ml	200	400	500	1,000	2,000	5,000	10,000
RR820B	2 x 5 ml (10 ml)	400	800	1,000	2,000	4,000	10,000	20,000
RR820L	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR820W	5 x 5 ml (25 ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000
RR82LR	5 ml	200	400	500	1,000	2,000	5,000	10,000
RR82WR	5 x 5 ml (25 ml)	1,000	2,000	2,500	5,000	10,000	25,000	50,000

High-throughput real-time PCR

Where throughput meets flexibility

Real-time PCR (qPCR) is a powerful technique for genotyping and gene expression analysis. Currently, qPCR experiments are becoming increasingly complex—involving an expansive and growing list of targets from a larger number of samples, all with more technical replicates. The SmartChip Real-Time PCR System is a complete high-throughput solution that enables an unrivaled amount of flexible assay and sample formats, allowing researchers to seamlessly switch between dispensing assay reagents and samples into blank chips, or dispensing samples into custom, preprinted chips without the need for revalidation.

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