Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:108576501-108576611 111bp TGCTGATGTGGCTCTTTACC CCAAGCAGGTTCTGGACTCT
Mut= 111 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGGTCCGGCTGAAGTGGAGGCACGGGTTTGGTAGGATGCTGTGTCCTGGTATATTCTGAGTCTGTATGCTAAGGAGTGGCCTGCTCCACTCATGCGTCCTAGGATCAGGTGCGTGCGTGCGTGTGTGGTGCTGATGTGGCTCTTTACCTCTGTCTAGGCGAGGGGCctgatttactggtgtacaacttggaccttggtgggcatctccgaatggtgaagagagtccagaacctgcttggtcactttctcatccatgggttccgagtgcgaccagagcctaaaggagacctggactctgaggccatgatagctgtgtttgggagcaagggcctcaaagttgtgaaagtcagctggggtcaaagccatcttcgggagctctggcgctctggcctgtggaacatgtccgactggatctgggatgtccgctggatcgagggtaacgtagccgtggccttgggccacaactcggtggtactgtatgacccagtgatagggtgcatgctgcaggacgtcccctgcacagacaggtgtaccctgtcctcagcctgcctggttggtgacacctggaaggaactgaccatcgtggctggcgcggtttccaatgagctcctgatctggtacccagccactgctttaacagacaacaaacccgtggcccctgaccggcgggttagtggccatgtgggtgtcatctttagcatgtcatacctggaaagcaagggcctgctggcaactgcttcagaagaccgaagtgttcgtctctggaaggtgggggacctccgggtgcctgggggtcgggttcagaatattggccactgctttgggcacagtgcccgagtgtggcaggtgaagctcttagagaactatctcatcagtgcaggagaagactgtgtctgcttggtgtggagccacgaaggcgagatccttcaagcctttcggggccaccggggccgaggtatccgggccatagccactcacgagaggcaggcctgggtggtcactgggggagacgactcaggcattcgactctggcacctggcaggccgagggtacccaggcttgggcgtctcatccctgtccttcaaatctcctagccggccaggtgccctcaaggctgtgactctggctggttcctggcgagtcctggcagtgactgatgtggggtccctgtacctctacgaccttgaggtcaagtcctgggagcagctgctggaggacaatcgctttcggtcttactgcctgctagaggcagctcctgggcccgagggctttggactctgtgccttggccaacggggagggtcttgttaaggtggttcccatcaacacccccaccgctgccgtcgagcagaaactgttccaggggaaggtgcacagcctgagctgggcccttcgtggttacgaggagctgcttttgttagcatcgggccctggtggggtgatagcttgtttggagatctcagctgcacccactggcaaggctatctttgtcaaggaacgttgccggtacctccttcccccaagcaagcaacgatggcacacatgtagtgctttcctgcccccgggtgacttcctcgtctgtggggaccgccgtggctctgtgatgctattccctgtcagaccatgtctattcaaaaagcctggggccggaagcaaggctattactgcagctgaggcacctggagctggtagtgggagcggtgggtctgagagtgtcccaacaggaataggccccgtctctacactccattctctgcatgggaaacagggtgtgacctcagttacctgccatggtggctacctatacagcacagggcgggatagctcctacttccagctctttgtacatggcggccacctccagccggtcctaaggcagaaagcctgtcgaggcatgaactgggtagctgggcttcggatggtgcctgatggaagtatggtcatcttgggtttccatgccaacgagtttgtagtgtggagcccccggtcccatgagaagctgcatatcgtcaactgtgggggagggcaccgctcctgggccttttctgatactgaggcagccatggcctttacctacctgaaggatggtgaggtcatgctctatcgggctctaggtggctgcatccggcccaatgtgattctccgggagggtctgcatggccgtgaaatcacatgtgtaaagcgtgtgggcacagtgaccctgggccctgaatttgaggtacccaacttggagcatcctgactccctggagcctggcagtgaggggcctggtctgattgacatagtgataacaggcagtgaggacactactgtctgtgtcctagcacttcccaccaccacaggctcagcccacgccctcacttctgtctgtaaccatatctcctctgtgcgagccctggcagtgtgggctgttggcaccccaggtggcccacaggatactcgaccagggctcactgctcaggtagtgtctgcagggggccgagcggagattcactgcttcagcgtcatggtcactccggacgctagcacccccagccgccttgcctgtcatgtcatgcacctttcatcccaccggctggatgagtactgggaccggcagcggaacaagcacaagatgatcaaggtggaccctgagacCAGGTAAGGCGGCCTCCCTGTCATGGGCGGGGCGTGAGAGGGTTATAGGAAAGTCCTGACTCCTGACTGCAGGCCCCACCTGACCGGTGTGTACCCCCTACAGGTACATGTCTCTTGCTATTTGTGAGCTTGACAACGATAGGCCTGGCCTCGGCCCTGGCCCCCTTGTGG
This mutation is a 2,470 bp deletion beginning at Chromosome 9 position 108,574,104 bp and ending after 108,576,573 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42316 | TGC TGA TGT GGC TCT TTA CC | Common | A | |||
42317 | CCA AGC AGG TTC TGG ACT CT | Wild type Reverse | A | |||
42318 | CCT GCA GTC AGG AGT CAG GA | Mutant Reverse | A | |||
42319 | Fluorophore-1 | CTT GGA CCT TGG TGG GC | Quencher-1 | WT Probe | ||
42320 | Fluorophore-2 | AGG GGC CAG GTA AGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42316 | 0.40 uM |
42317 | 0.40 uM |
42318 | 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 |