Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:87567771+87567864 94bp AAACTTGGTTTCCCACTTGC AAGGCAGGATAAAGGGCTCTG
Mut= 90 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTTTGGGGATCTTGTCACAGATGCCAGGGATACATGCCAGGGTGGTCCAGAGGCCAAGGGACAGTGAAGGGACAAGAGGCTCTAGAGTATTGGTACTAAAACTTGGTTTCCCACTTGCCAGGAATGGCCTCCTATTTAGAGacagtgggtagaacccccacaggatggctgcagagccctttatcctgcctttacagaaaaagaaggaagagacaaagagtacagaagagaaggcaggaaagaaaaacgtaggcgctaagaagaagctagaaaaagtcagccgcaaagtgaccttccacaaggtaacacctctccccctcccccacagctgctcctctactatggtgtctgccacccctctctgaaccataatgacaccctaagagatgagtcttctcacgcgcgcgcacacacacacacacacacacacacacacacacacactccttgcagcttccccaacaacctctgctttcctgcagtccagtttaagggtgagcgtctcacattcctccacccacgtccctccatactccatgttaccagggcccagagaaccatgggaacactcagtcctgtacctacagggtatcccagacctgcctgcgagcatagactgcatgtaccctcatgagacaaggagaaataaaagtgaagtctcagggagccagcatggaagacacttctgctccctgaaactcttcctgggctcagaccaggtcctcgagcaagctggagaaatttctcagtttgggtttagaggatgctgagcaaggtcctctaagcagagcataacttctctcctgctttccaagggctacgtacgtcctgtcaagaagaaggaacccaccatttaccacaagagaaatgaaatagacctaaagagcaagcgtctgagcatgatcactgccaacttcagggatagcaataagaagtctggtaaggaaaatacacaggccctgggataccagaactgacctcccttgtccaatttctaagagataatggaagagctgcagcatccatgctgatccaacctgtgtacataggagccaggagccagcatgcagggttgttagggaatcacccagGGTAGCATTTGACAATCTGGGTATGCTGTGGATAGGAGGGGAGTGGTTGTTGATCTCTACATTGTCAGGAATCCAGCACAGTGTGATACATGCTCCTGAACTATTCCAGTGATGTCCAAATTCTTTAACTCAAACACTAT
This mutation is a 950 bp deletion beginning at Chromosome 8 position 87,567,814 bp and ending after 87,568,763 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45262 | AAA CTT GGT TTC CCA CTT GC | Common | A | |||
45263 | AAG GCA GGA TAA AGG GCT CTG | Wild type Reverse | A | |||
45264 | ACC ACT CCC CTC CTA TCC AC | Mutant Reverse | A | |||
45265 | Fluorophore-1 | CAG TGG GTA GAA CCC CCA C | Quencher-1 | WT Probe | ||
45266 | Fluorophore-2 | AGG GTA GCA TTT GAC AAT CTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45262 | 0.40 uM |
45263 | 0.40 uM |
45264 | 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 |