Home › Cell Biology › How to Design Hydrolyis Probes for qPCR
Steps
  1. 1 Understand probe melting temperature requirements 00:30
  2. 2 Determine appropriate probe length 00:44
  3. 3 Avoid five-prime terminal thymidine 00:54
  4. 4 Design probe spanning exon-exon junctions 01:04
  5. 5 Validate probe sequence with BLAST alignment 01:17
Cell Biology Bio-Rad Laboratories

How to Design Hydrolyis Probes for qPCR

Protocol
Difficulty
intermediate

Steps

1
Understand probe melting temperature requirements

Learn that hydrolysis probe melting temperature should be 8-10 degrees Celsius higher than primer melting temperature to ensure optimal performance in qPCR.

▶ 00:30
2
Determine appropriate probe length

Design probes shorter than 30 nucleotides for most applications. For longer probes exceeding 30 nucleotides, consider incorporating an internal quencher.

▶ 00:44
3
Avoid five-prime terminal thymidine

Ensure your probe does not have a 5-prime T (thymidine) as terminal nucleotide, since T residues are poor quenchers and cannot effectively suppress fluorescence after hydrolysis.

▶ 00:54
4
Design probe spanning exon-exon junctions

For gene expression studies in eukaryotic organisms, design the probe or one primer to span an exon-exon junction to prevent amplification of contaminating genomic DNA.

▶ 01:04
5
Validate probe sequence with BLAST alignment

Run the finalized probe sequence through a BLAST alignment tool to confirm it does not bind to any other sequences in your genome of interest.

▶ 01:17
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