Home › Molecular Biology › How to design primers for real-time PCR (qPCR) for gene expression? Easily and quickly. PrimerQuest.
Steps
  1. 1 Understand qPCR primer design overview --:--
  2. 2 Access PrimerQuest tool and create account 00:29
  3. 3 Input gene sequence and select qPCR option 00:57
  4. 4 Review and download primer design results 01:24
  5. 5 Select alternative probe-based detection method 01:52
  6. 6 Customize primer design parameters 02:15
Molecular Biology YouTube (Curated Tutorials)

How to design primers for real-time PCR (qPCR) for gene expression? Easily and quickly. PrimerQuest.

Protocol
Difficulty
intermediate

Steps

1
Understand qPCR primer design overview

Learn that real-time PCR (qPCR) is used to validate gene expression and understand the importance of designing appropriate primers for your target genes.

▶ --:--
2
Access PrimerQuest tool and create account

Navigate to the Integrated DNA Technologies website, locate the PrimerQuest tool under Tools, and create a free account to sign in.

▶ 00:29
3
Input gene sequence and select qPCR option

Insert your coding DNA sequence (CDS) or cDNA sequence into the tool and select the qPCR primers option for intercalating dye-based detection.

▶ 00:57
4
Review and download primer design results

Examine the multiple primer sets generated by the tool and download the results in Excel format for further analysis.

▶ 01:24
5
Select alternative probe-based detection method

Choose the TaqMan Pro-based option to view primer sequences along with probe sequences for hydrolysis probe-based qPCR experiments.

▶ 01:52
6
Customize primer design parameters

Modify default settings such as primer length, melting temperature (Tm), GC content, amplicon size, and target sequence region to match your experimental requirements.

▶ 02:15
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