Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:80547672-80547808 137bp GGATTGCTCACGTACCCAAC TGCTCAAGGTCAGGTACAACA
Mut = 142 bp
Wt = 137 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTGCTGATCCTCCGTAGGGCTCTCATTGGCAGGACCGCgtagggtctggcggcaatgaggctgggtgggaaggcaggggtgtgacccaaggaggcaccagagtgaggagtgcatgtgtgttttcagccacccaaggagaaggctgaggagaagacacatgcgggcctgaccactttgttcccggggcagaagaggagggtctcacacctttgcaagccaggcaaggaggtaagacccagatatggggatacagggcgggcctctggaggaaggagatttgccaccacagttgggcaaaggacccctgctgtcggcctttggtaagctggtatctggggcttcatgtcccatccagatctgggcacaggaacagtgcacggaggggtggataaatccccatgggttttagagttgagtgggcagtccaacctcagagcccaggctatctcagttgtggccggaaggtggtgaggatagtaggggaggcctgtgtggggtgtggtagcaagcaagggctccacaaacatccctggtgcagtctccgaggagacatgagcgcagacagagtactggtgggcagaggaacatggaagaggagcttctgcagcctcctcaccatgcttgtctggagtgctctggacgggggctagctctaggatctgtgctctttcagtcagaggccccaaagaggacccccgtggctcccccagcacgccccccgactgctcaggaggtgtgctaccggcgagcacagctagcacagagggactcggccagttggctacaggccgccccgcggccaacagagaggctctcctccgtgcacatctcagcgcctggggagaagaggaggattgctcacgtacccaacccccgactggctgccggtaagtcctgaccctgccctttcttggaccccttgccttggctcgtgcagtatgagaggaagcagcgcTCGGAGAACTTATGTTGTACCTGACCTTGAGCACCCTCC
This mutation is a 917 bp deletion beginning at Chromosome 10 position 80,547,705 bp and ending after 80,548,621 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53071 | TGC TCA AGG TCA GGT ACA ACA | Common | A | |||
53072 | GGA TTG CTC ACG TAC CCA AC | Wild type Forward | A | |||
53073 | GAC ACG CCC ACT TGT CCT | Mutant Forward | A | |||
53074 | Fluorophore-1 | CGG TAA GTC CTG ACC CTG CC | Quencher-1 | WT Probe | ||
53075 | Fluorophore-2 | CTC ATT GGC AGG ACC GCT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53071 | 0.40 uM |
53072 | 0.40 uM |
53073 | 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 |