Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:83300864-83300969 106bp CCATCTATCCATCCATCCATC CAGGAGGCAGAAGGTGGTAG
Mut= 106 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCCTCACTCCACACCTGTTGACACAGATCAGGGCATTTGGAGAGCCCACTGTCTTAAGTGGTAATAAAACTAAAACCCAGGAGCCCCTTCAAAGTGAAAGCAGTTCAGTAAACCATACCCAGCACCctaatgatcttgtatgtaatgattaactaatgatagtttaatgttttaaaaattactaatctggatttcacaaaaatgtttttaggttcaggccacagtggttggctttttagcagctgtggcagccatcatactgggctggattccagaagggaagtattacctcagccattccatacttctgtgctccagcagtgtggcaacggccttcatcgcatctctactgcagggtaaggggatctgtgaaggtcgacttggcagttcttgacatttcaataaaaggattgatttcccaggatggcaacttttaaaatatttgttcataatgaaaatggtagcaaatttccacataaactttaaagagccttagttcaaatttatttctctgtctcatctacctatctacctatccatccatccatctatccatccatccatccatctacttgagttctttgtatacagccCTAGCTGTGCTGGACCAGGCTGGCCTCAAACTCACAGAGGTCTACCACCTTCTGCCTCCTGAGTACTGGGATTAAAGGCGTGCACCACCACCTGATTAATTTTATTTGTTTCATTATTTACAGACACAGTGGAGGACAGGCTTTTTGAGCTCGGCC
This mutation is a 467 bp deletion beginning at Chromosome 10 position 83,300,925 bp and ending after 83,301,391 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41247 | CCA TCT ATC CAT CCA TCC ATC | Wild type Forward | A | |||
41248 | CAG GAG GCA GAA GGT GGT AG | Common | A | |||
41249 | AGC CCC TTC AAA GTG AAA GC | Mutant Forward | A | |||
41250 | Fluorophore-1 | CAT CTA CTT GAG TTC TTT GTA TAC AGC C | Quencher-1 | WT Probe | ||
41251 | Fluorophore-2 | CCC AGC ACC CTA GCT GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41247 | 0.40 uM |
41248 | 0.40 uM |
41249 | 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 |