Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:123840401-123840500 100bp TCTCCTTCCCCATTTGAGTG GCTTTGACTCTTAGTCTCACCACA
Mut= 96 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATACCAACTATTTGTTGACTTGCTGTATATACATAGTGCTAGGTTGGTGGTAGATACAGGGATGAATTTTTTAAAAAACAAAACAAAAAACCCCACCCGTGCCCAATCAGCAAGCTGTTTCAGTGTCTATGGGGACTTAGTACACTTGACTCTCCTACCTGaatagttgaatgtcctgtgtgtttccaaggttacacttactccttgtgcttcttggtgttatttctacttctttctcagacttacttttcagtacatctaagatgcgtgtcaattctagggctgttaaaatgtgaagtagacaaatgtgtctctctagttacgtgttcctatttgttgtcttaaagatgcagcgacagaactaagccatgaatttaaggaaggcggagagccaatcacagatgacagcaccagtttgcacaagttttcttacaaacttgagtatcttctgcaagtaagtggcccctgtcatgcactggttttgtcagagttgtgtctccttccccatttgagtgactgttagcagtttccagggtgTGGTTCGGAAAGCACAGTGAAGCAGTTAAGTCCTTGTGGTGAGACTAAGAGTCAAAGCAGTGCCAGGAGCTCAGTCCTTCCACGTGAGTCAGGGTGTTCTGGGAACTGAAAACATTCTTCTGAGACTTAAGCCATCGTAAGGCAAGTCACAGGCCTTTCCAGCATTACACCCTTTGAGAACTTGTCGA
This mutation is a 376 bp deletion beginning at Chromosome 9 negative strand position 123,840,459 bp and ending after 123,840,834 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38712 | TCT CCT TCC CCA TTT GAG TG | Wild type Forward | A | |||
38713 | GCT TTG ACT CTT AGT CTC ACC ACA | Common | A | |||
38714 | TGT CTA TGG GGA CTT AGT ACA CTT G | Mutant Forward | A | |||
38715 | Fluorophore-1 | ACT GTT AGC AGT TTC CAG GGT G | Quencher-1 | WT Probe | ||
38716 | Fluorophore-2 | TCT CCT ACC TGT GGT TCG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38712 | 0.40 uM |
38713 | 0.40 uM |
38714 | 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 |