Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 74 bp
Wild Type = 86 bp
wt sequence with deletion in lower case
ATGACAACAACAAACCTGTGTTTTCCCTACTTGCTGTGATAAAGGCAGAGAACAGTATCTGCCTGCACCTACAAATACACTTTCCTGTATCATCCAAAGCCATTTAGAAAACCACATGAGAACATTTCTAACACGCTTTCCTTTATAACACAATGCTGCAATAGCTTACCCATTtacagcactgtagagatcatttcatagttaatcccattaatactgaaaataatatctaatagtgcttctaagttggaagtcatatttcagacacttactaggaaaaatgagtagtgcatcagaactgctagtcactgctgttgtccagagcagtgtctggacaaaatagaactcagtaaatgcttgttgaatgaatcaatgggcttttcttttcaggcctgtattgatgaaaatttggacatggtgaagtttctggtggagaacagagccaatgtaaaccagcaggacaacgagggctggacacccctgcatgcagcagcttcctgtggttatctcaacatagcagagtgagtgagcggcgttgtgggtttgatgcacattcctcctctgcttcccagaaatggaatttcacaaatagttataaatttctctaaattacaaaagcagaaaattgactattaagagctagctgacactgtgctttgtatctcaatgcacagttcttagctccaatttttgtttgcttctgtttttgtatagttcataatttctggactattcagaggcagcatcagcctggcATGCTGTCTGGCTACTTTCCAAACAGCAGAACACAAGCATGGGAGTTGTAGTTTGTTTGTTTGTTTTTTTAACTTTTCTGTAAACCTGCTTTTTGTTTGTTTGTCTTTATAAATATTATTGCAGTAAATAGCAAATTCTTAGCTGGAACAGATGGAACCAAGGCAAAG
This mutation is a 581 bp deletion beginning at Chromosome 1 position 134,902,144 bp and ending after 134,902,724 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44171 | GGT GGA GAA CAG AGC CAA TG | Wild type Forward | A | |||
44172 | TGT TGA GAT AAC CAC AGG AAG C | Wild type Reverse | A | |||
44173 | CAA CTC CCA TGC TTG TGT TC | Mutant Reverse | A | |||
44174 | CAC AAT GCT GCA ATA GCT TAC C | Mutant Forward | A | |||
44175 | Fluorophore-1 | ACA ACG AGG GCT GGA CAC C | Quencher-1 | WT Probe | ||
44176 | Fluorophore-2 | CTG TCT GGC TAC TTT CCA AAC AGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44171 | 0.40 uM |
44172 | 0.40 uM |
44173 | 0.40 uM |
44174 | 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 |