Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:58536148-58536283 136bp GGGGTATAGTCAACAAAATCTCCA TGAAAACCATTGCTATTGACC
Mut = 136 bp
Wt = 136 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCAGTAGATCTATATCTACTCATTAAGCAGATGAAGGCAAATAGAAGGGTGGGGTATAGTCAACAAAATCTCCAGATTAAATAAAATGTTTTATGGCTGGAGttgtgggaggatcaagttctcattttaaatgttaaaataaagttttggagtatgcattttttaggtcaatagcaatggttttcatctcaaaatgttcagaggagccatttaattaacaaatcttcttgcattgcttgttctgggccctgctgtgcttatcatccatgcagaacaaaattcttccttttttatgcttgaatactttctttctccacagacatttccactgggcctaggagatgggcaattctattcctggacagatggtctgagaagaaaagactggcatgactatgagagcattcagagagatgctttgcgctcaggtatggattcagcatcagacagagtggtgctgttacttcagtgtttggtttgtccctgctaagttcttatgtccgtttttcttctgcatgcactaaagttgtctgtcttatgggaacattggtaatcaacaagGGACAGAAACAGATGAATTCAAACAAAACAGACAAAACAAATAATGAACATCCTTTCCTTGGCTGCAGAGGAGGTCATTGCAAATGTGCAAATCTATATTGTCTTAGATTCCTTTTAAAATAATT
This mutation is a 459 bp deletion beginning at Chromosome 8 position 58,535,773 bp and ending after 58,536,231 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50637 | GGG GTA TAG TCA ACA AAA TCT CCA | Common | A | |||
50638 | TGA AAA CCA TTG CTA TTG ACC | Wild type Reverse | A | |||
50639 | TTT GCA ATG ACC TCC TCT G | Mutant Reverse | A | |||
50640 | Fluorophore-1 | TGG GAG GAT CAA GTT CTC ATT TTA AAT | Quencher-1 | WT Probe | ||
50641 | Fluorophore-2 | CTG GAG GGA CAG AAA CAG ATG AAT | Quencher-2 | Mutant Forward |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50637 | 0.40 uM |
50638 | 0.40 uM |
50639 | 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 |