Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:127888946+127889051 106bp GGTCTTCAGGCATCTTGCTG TGCATTCGACAGTTATCCTCT
Mut= 106 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTTATTTGTCTGTGTTTATGTGTGTGTGTTTGTGTGTGTGTGCACAGACACGCATGCGTGCATGTATACATGCTGATGTGTGTGGAGGTTAGAGGACAACTTGGAGGAGTCAGTTCCCTCTTTCTATGTGGTTTCCAGGAGTCAAACTTAGGTCTTCAGGCATCTTGCTGACTCCAAATagccactgtgataagtcatttattctcctgaaattatttttttgcctttatcatgtagaggataactgtcgaatgcagtgctatgattaatgttagaaaagagacaaaaatcattgcatagtaaacactatgatgactgttgagtaagctggctgttgtttctttctcttgttaaatgcagggtatatttacttctggcacatgggaagagcgatcagatgaaatttcctttgctgacttcaggttttcagtcacacatcactatcttgtacaagagtctcctgataaagaaagaaaggatgaagaattggagggtatggggtttttgtttttgttttgtggtttgttttttttttggagggggatcatatttgtacaatctttaaagagtctaatttttagttattagtttagttctcataactcaataatacccataggaagcatttttttttgtaaccacatttgaaatagcttatgtgctagtactgcatctgaagAAGAGGAGAAGCCACAGTGACGAGCACTTTAGACTTGATTGAACCAGCAGATATTGACTGAGTGGCTCATTTGGTGATTGGTGGTAGGTCATGTAGTCTACTCTGAGATGCCCTATGTGTAACAGTGAGCAGGGGGTTTCAGCCGCTAGTCAAACAGTAGTCATTAGTCCTCCTGTTGTC
This mutation is a 500 bp deletion beginning at Chromosome 1 position 127,888,975 bp and ending after 127,889,474 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46807 | GGT CTT CAG GCA TCT TGC TG | Common | A | |||
46808 | TGC ATT CGA CAG TTA TCC TCT | Wild type Reverse | A | |||
46809 | TCA CCA AAT GAG CCA CTC AG | Mutant Reverse | A | |||
46810 | Fluorophore-1 | AGC CAC TGT GAT AAG TCA TTT ATT CTC | Quencher-1 | WT Probe | ||
46811 | Fluorophore-2 | CCA CAG TGA CGA GCA CTT TAG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46807 | 0.40 uM |
46808 | 0.40 uM |
46809 | 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 |