PCR Multiplex PCR - Mammalian DNA

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. Multiplexing such a reaction amplifies the design challenges where one target requires 3 primers, which should be exclusively bound nowhere in the template DNA or to each other. Similarly, two targets require 6, three require 9, and so on. Each amplicon needs to be either a different size (for gels) or labeled with a different fluorescent tag that is spectrally distinct from the others in the reaction. Further complicating this, different targets in the reaction can compete with each other for resources and causes more challenges in the detection of amplicons. However, with proper primer designing, their validation, optimize quality and concentration of the enzyme and buffers certainly lead to a successful multiplex PCR reaction.

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1 year ago

1 year ago by R. Verma India

Multiplex PCR and qPCR

I have been doing multiplex PCR and qPCR separately. Is there a way to combine them in a single reaction using Sybr Green?

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2 years ago

2 years ago by Roland Lafontaine France

My multiplex PCR has decreased in sensitivity

Hello there! My multiplex PCR has decreased in sensitivity even though I have not changed any of the parameters. The materials are in good condition and the PCR machine is well calibrated. I have tested multiple samples but one of my targets has decreased in sensitivity.

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Found 6 matching solutions for this experiment

Phusion U Multiplex PCR Master Mix

Thermo Fisher Scientific

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Ion AmpliSeq™ Library Kit 2.0

Thermo Fisher Scientific

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Follow manufacturer’s instructions
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