Discover Ways to Increase Your qPCR Productivity

Increase your qPCR productivity with tips on assay design and more

Below you will find tips for qPCR assay design as well as videos about best practices for real-time PCR assays, including how to measure PCR efficiency, baselines, and thresholds; the best quencher molecule; and the purpose of ROX dye in your assay.

Prepare your sequence with bioinformatics by masking SNPs and repeats
Check that your target sequence is unique in the genome and transcriptome (e.g., BLAST)

Use an assay design tool like Primer Express or OligoPerfect
For mRNA transcripts, one of the primers or probe for TaqMan should span exon boundary
Keep the GC content in the 30–70% range
Do not overlap primer and probe sequences
Probes should have a Tm ten degrees higher than primer

Order custom primers and probes using our ordering tool.

Detect your positive controls
Ensure no signal in NTC (no-template control)
Single peak in melt curve with SYBR primers

Ideally efficiency is 100% +/- 10%

Step 1 : Perform bioinformatics

Prepare your sequence with bioinformatics by masking SNPs and repeats
Check that your target sequence is unique in the genome and transcriptome (e.g., BLAST)

Step 2 : Design your assay

Use an assay design tool like Primer Express or OligoPerfect
For mRNA transcripts, one of the primers or probe for TaqMan should span exon boundary
Keep the GC content in the 30–70% range
Do not overlap primer and probe sequences
Probes should have a Tm ten degrees higher than primer

Step 3 : Order primers and probes

Order custom primers and probes using our ordering tool.

Step 4 : Validate your assay

Detect your positive controls
Ensure no signal in NTC (no-template control)
Single peak in melt curve with SYBR primers

Step 5 : Check your assay efficiency

Ideally efficiency is 100% +/- 10%

Know your target but don’t want to do all the above work? Use one of our more than 28 million pre-designed TaqMan Assays. These assays are designed with a validated process and come with the TaqMan Guarantee.

Have a target not found using the pre-designed assay search tool? Use our Custom Assay Design Tool for custom assays designed using the same process as our pre-designed assays.