Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:51861204-51861309 106bp GCCTTGGATGGAGATTTCG CAGCCTGGGAACTGAGAATCTA
Mut = 99 bp
Wt = 106 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCTCATGTCCAATCGGCTCGCGCCTCGGGATGTCAGGGCGGAAGCGGCcgccatggagccgcttgtgcagcaaaccgagcgattctccgagctgttggccgtgtcgtgtgggtcgctggtgagcacgtgggacgccgaaaaggtgcgcagggcgctgcagtgggcgcgttatctgctccacgtgtaccgacgcttcgcgggccgcggccgcgtgcgggaggcgctcgagaggcggctccccgcgcggggagggccgctcgggctgcgcagcttcgcggcgctggagtccggggacgcgcggctcgccctccgcctgctgcggaaccgcgcgctcgcgcccgccgccgcccgcgcgctgccgagcttgctgttcccgggccccgccgcggaccaccgagacgacgtgccgcagtcgcgcctggtcctcctggcgcgccgcggaagcgcgctgcgcctgctgtgccgcctcggcggggacgcgcccaggagcgcgctgctgcgcacgcacgcggagctgctggacgcgcgcctgcacgagctgggcggggccgactccgcggccgcgcgcaaactcctggacacgttgtggacgcgcgggccgcgggagcacgtgctcgacgtgactgccgaagcgctgctgctacgcgaggaagacccggagccagcccaggccacggaccctgcgggtgcggatgagacacagaaactactgcgctggcttctcgaaagtcccgaggtattggccgctttttgccgccatctgccagccaagcgcctggcctccgtggcgggctgccaccacgcactgtctcgcgcctacctggacctgctcacaacgtgggccacgcggctgcactatgatctgcaaaagggtgcctgggtccctacacagatggaggacatgccctgggaagagctgtgcctaaggctgcagagcttgtgccatgcccagccatttctgcaggaagaggtgctggtgaccctaaggtcccgcaaagccttggatggagatttcgaggttcctgggatgagcatctggacagacctcctggtggtgcTTGAGTGTGGGATCGTGCTAGAGTAGATTCTCAGTTCCCAGGCTGGG
This mutation is a 1010 bp deletion beginning at Chromosome 7 position 51,861,249 bp and ending after 51,862,258 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51862 | CAG CCT GGG AAC TGA GAA TCT A | Common | A | |||
51863 | GCC TTG GAT GGA GAT TTC G | Wild type Forward | A | |||
51864 | GCT CAC GCT CAT GTC CAA TC | Mutant Forward | A | |||
51865 | Fluorophore-1 | CCT GGG ATG AGC ATC TGG A | Quencher-1 | WT Probe | ||
51866 | Fluorophore-2 | AGG GCG GAA GCG GCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51862 | 0.40 uM |
51863 | 0.40 uM |
51864 | 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 |