Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:22202154+22202249 96bp GCAGAGAGGGAGCAGTTTCT GCTGCGTTACACTCATCAGAAG
Mut = 93 bp
Wt = 96 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAACACTTGCCGGGCAGAGAGGGAGCAGTTTCTTTAAGGATTACAGCATTGTTGTTTTTAAGTACctcctagggaacagcgctctatcttctgatgagtgtaacgcagctctttttctgtgggtttcctctaggagtgctgcatggccattcccctgtctcagattgtgttcatcgaggagcaggcagctggaatcgggaagaggtgaggtctaacagtcctcagtcacaaaaccatttcctgtcactggtcagtaggccttttaagggtgtgtttccaccttatatccattttgaagtctgaataaagtggtaaggcttgctgtaatatccttgcatcataataaatgatggggccctgctcttacCGTATGTATTTCCACAGCACGCTTGTCGTTCATGTGTGTGCATTATTCAGTATTTAAACTCAGGAATGCCTCAGCTGAGGGAAAGAACTATTAATGTTGATAACTTATTGGTAACC
This mutation is a 302 bp deletion beginning at Chromosome 8 position 22,202,206 bp and ending after 22,202,507 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51242 | GCA GAG AGG GAG CAG TTT CT | Common | A | |||
51243 | GCT GCG TTA CAC TCA TCA GAA G | Wild type Reverse | A | |||
51244 | GCA CAC ACA TGA ACG ACA AG | Mutant Reverse | A | |||
51245 | Fluorophore-1 | CTC CTA GGG AAC AGC GCT CT | Quencher-1 | WT Probe | ||
51246 | Fluorophore-2 | AGT ACC GTA TGT ATT TCC ACA GCA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51242 | 0.40 uM |
51243 | 0.40 uM |
51244 | 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 |