6Select primer location relative to gene ends05:58
7Verify primer specificity using BLAST06:32
Molecular BiologyYouTube (Curated Tutorials)
The Features Of A Good qPCR Primer Pair
Protocol
Difficulty
intermediate
Steps
1
Understand PCR product size requirements
Learn that optimal qPCR products should be between 70 and 200 base pairs, with 150 base pairs being ideal. Products outside this range will have low primer efficiency.
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2
Verify primer length specifications
Ensure each primer in the pair is between 18 and 22 base pairs long, with 20 base pairs being the optimal target length.
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3
Check melting temperature and GC content
Confirm that each primer has a melting temperature between 59 and 65 degrees Celsius (ideally 62ยฐC) with both primers matching within 1 degree. Verify GC content is between 50-60% for each primer.
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4
Identify GC clump and sequence repeats
Confirm that each primer contains at least one G or C base in the last 5 bases to ensure complete binding. Avoid primers with repeats of the same nucleotide sequences.
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5
Design primers spanning different exons
Position primer pairs to bind on different exons separated by at least one intron, or use exon-exon spanning design. This prevents amplification of genomic DNA contamination.
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6
Select primer location relative to gene ends
Design primers near the 3' end of the gene if using oligo dT during reverse transcription, or near the 5' end if using random hexamers. This ensures cDNA template availability.
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7
Verify primer specificity using BLAST
Use NCBI's Primer-BLAST tool to confirm primers bind only to the intended target gene and not to unintended templates elsewhere in the genome. Check for high complementarity matches that could cause non-specific amplification.