Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 85 bp
Wild Type = 88 bp
Genomic sequence with deletion in lower case
AAGCAATCTAGAGCTTAAGCCTAGGGTGGCAGATGTGTGGAAGAACACAGACACAGTTGTGCAGAGCCTGGGAAACGGCTTGGGcttccagggaagaggtttatgttatcgttgtttgggttgggttgtttatttctgggggctgggggagggaaggtatgtatgttttgttgtttagtatctcatgtagccaggatggccttgaactcactatgtagctcagactgacgtggaattccaggttctctctttactccccacactggtagctgtgcaccataaaacctggcttatactttgtaaaatcccaatattctcttgcttgctttcagcacccttatcacatgtgtggattctgggatcctcagagtctggtgtgaaaatgacaaagaggcgtcctctgacccagtaaggtttacctgtcccggtggttgaaaggatgggagctagggtaggggtctgatggggaatttggtaaaggaaagattaagggctttgagactaactcatctctggttattttcagctcttggaactgaaggtgggtcctggggtgtgtaggatgcgccaagatcccacacatacacatgtggttgccacatgtgggaaagaaaatgctttgaaagtatgggacctacaggggtctgaggagcctgtattcagggccaaaaatgtgagtgagtgatgaagggttggggagatgctatgtttgattgaggttggggaagtgaaagccactgacatgacctagatggctttgggggtatggaggattggaggaaacaggtcaggcttatcaaactttgtgcccatatgtaccttccacaccaggtgcggaatgattggcttgatctccgagttcctatttgggaccaagatacacagttcctccctgggtcacagaaacttgtcacatgcacagggtaccaccaggtgaggctctttttactcccggattgaagtagcttgaattcagaaaaacaacaaaaaaagtgtctcctgatagagtaggtaggttgcatggtgtaactctgcctgggttatcaaaggagctcatactttcttagtcaacaaagCCCATGGCTGAGCCTACTACTTTCCCCTTACTGCATTCCAGGGTGGAATTCTGAACTCTGGGTTTCTATTTCTCCTTCCTAGAAG
This mutation is a 992 bp deletion beginning at Chromosome 19 position 8,737,406 bp and ending after 8,738,397 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44407 | TGT GTG GAA GAA CAC AGA CAC A | Mutant Forward | A | |||
44408 | GCA GTA AGG GGA AAG TAG TAG GC | Mutant Reverse | A | |||
44409 | CGT CCT CTG ACC CAG TAA GG | Wild type Forward | A | |||
44410 | TCC TTT ACC AAA TTC CCC ATC | Wild type Reverse | A | |||
44411 | Fluorophore-1 | AGC CTG GGA AAC GGC TTG G | Quencher-1 | MUT Probe | ||
44412 | Fluorophore-2 | AGG ATG GGA GCT AGG GTA GG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44407 | 0.40 uM |
44408 | 0.40 uM |
44409 | 0.40 uM |
44410 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |