Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:37683015+37683094 80bp ACCAGTTTCAGTCTTTACTGTTGG GAGTGTTGACTTGTAGGAAATAGTCTC
Mut= 82 bp
Wt= 80 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGAAATTAAAACACTAAATACAGAATTTACTTCAATAGATATGTAAAATGTAAGGATCATAATCCACTGAATACTAAGACTGAAAATCCACTTGCTACTTGGGATATGACAGTCGGGCTTGAGTTTCTAATTGCCATACATGAAGACATTGATGGCATTTGTTTCCAAAACTGGAAAAAAAGAAAAACGTGCGCcttgtgaattgggaaggataatatataatgatgaaaacattcatgaaatgataataagcaaaataaaatatctaaaccaaaggggaaaaatgtgaatacctttgctgtaaattaaagtcaaaacaaaagctttgataaatagctgcacgatacgcagttttacaagacaaccattaggcgacgctgttgaccaaagggaacagcagggttctccgttccgcagggtccagcgagggtttgcttgctttttccagcacccatgagaacatccgtccggttcctgtgccctcttcaagcgccgggcaaatcctgccctcaaatcacaaacatctagggcactagctgtctttttgaaatcccagagaaacctcgaaaagaagctacctggcaagctcagaacggcagagcaaccccagcggctttggaacgatggaaaatcaggtattgcttcacttcctggtgcctttgttctgcggcgccctctcccagcccatccactactccattccggaggagctagccaagggctctctagtggggagtctcgccaaggatctgggactcagtatccaagaattgccagctcgaaaactgagagttagtgcagaggattatttcagcgttagtgcagagagtggggatttgttagtgagcggcaggatagacagagaaaagatttgtgggaggaaatctgagtgtgcactggaatttgaatctgttactgaaaacccgatgaacattttccacgtaacggttgccatccaagacattaacgataatgcaccacatttctttgggaaaagcattgaattggagatctgtgagtcagccttagcaggggcaaaattccctctggactctgcgcgagatgcagatgtgggaagcaactccctgaagatgtacactgtcagccccaatccacacttctccttgtcaacgaaggaaagtcctgatggaagtaaatacccagaactgctgctggaaacccctctggacagggagcatcagagctcccatcacttaatactgaccgccatggatggtggagacccgcccctaagcagcaccacccaggtctggatcaaagtcaccgacgccaatgacaatgctccagtgttcagccaggacacttacaaagttagccttcgtgaaaacatacccccaggaacgttagtgctgcaagtgacagcttctgatcaggatgagagtattcacggagagatcacctatgccttccttaatgccccagcaagtactaaccttgtcttcaatctcaatccaaatactggagctatcacaactaatggtacattggattttgaagaaaagaatagatacacattgggtgtagaagccaaggacggaggggtgcacacggctcactgtaatgttcagatcgaaattcttgatgacaacgacaatgccccagaggtgacattcatgtccttttctaaccaagttccagaggattcagtccttggaacagtaatcgccctcattaaagttcgagacaaagattctggacaaaatggtttggtggcatgccatattcaggaagaacttcccttcaaattacaacccacctccaagaattattacaagttagtgattgacaaggccctaaaccgagaacagaccccagagtacaacgtcactatcacagctactgacagtggcaagccctcgctttcctccaagacatgtgttactgtacacatcacagatatcaacgacaatgcaccggtttttcaccaggcctcctacttggtccacgtggctgagaacaacccacctggagcctctattgctcaagtcagcgcctcagatccggatttgggatccaatggcctcatttcctactccatcatagccagcgacctagagccaagggcactatcgtccttcgtgtccgtgaaccaggacagtggagtggtgttcgcgcagcgcgccttcgaccacgagcagctgcgttccttccagctgacactgcaggcgcgggaccacggatcgcccacactcagtgccaatgtgagcatgcgcgtgttggtgggtgaccgcaacgacaacgcaccacgcgtgctgtatcccacacttgagcccgacggctctgcgctcttcgacatggtgccgcgcgctgccgagcccggatacctggtcaccaaggtggtggctgtggacgcagactccggacacaatgcctggctgtcttaccacgtgctgcaggccagcgatcccgggctcttcagcctgggactgcgcacaggcgaggtgcgcacagcgcgtgccctgggagacagggactcagcgcggcagcgccttctggtcgctgtgcgtgatggtggacagccaccgctctccgctacagccacgctgcacctcatcttcgctgacagcctgcaggaggtcctgccagacctcagcgatgaccctctaccatctgaccctcagagtgagcttcagttttacctggtggtggccttggccctgatctcagtgctcttcctcctggcggtgattctggctattgccctgcgtctgagacactcctccagatcagctgcctggggctgctttcagcctggcctcagcctgaagtctcaacctgtggttcctcccaactacaatgaggggacattaccctattcctataatctctgcgttgcttcacattctacgactatggcagggtttaattttcaccacgtggcaccagaggttgctccccctcaggatctcctgtgcgatgatccttctgtggttgtatgtggcaataatgaagatccccaagtctcggacgactctgctttccaggtaagtttctacagagcaacttaaatgttatttgctgtatttacttctcaatcgtgcacaaacctgtcaattccgaacatgttttcatagtttgggggatatttagtggaagacgtgtcgtaggtgcgtaaagattggctgttggttgctcagtcatcttggaaatgacctgattaccagtttcagtctttactgttggtgtttctgtgtggctagcaaatCTTGCCGGAGACTATTTCCTACAAGTCAACACTCAATAATATTAGTGCCCTTTCTTACTTAATTCAGGAGACTGAATTTTAAAATAATCTTTAATTCTTTATAAGCACATTGATATGTCTTTCTGCCTAAAGTTTATGGTCAATTACCTTTCTTGCTTGAACAACTTTTGTTTGTTTGTTTGTTTGTTTGTTTGTT
This mutation is a 3032 bp deletion beginning at Chromosome 18 position 37,680,029 bp and ending after 37,683,060 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38898 | ACC AGT TTC AGT CTT TAC TGT TGG | Wild type Forward | A | |||
38899 | GAG TGT TGA CTT GTA GGA AAT AGT CTC | Common | A | |||
38900 | CAT TGA TGG CAT TTG TTT CC | Mutant Forward | A | |||
38901 | Fluorophore-1 | TGT TTC TGT GTG GCT AGC AAA T | Quencher-1 | WT Probe | ||
38902 | Fluorophore-2 | AAA ACG TGC GCC TTG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38898 | 0.40 uM |
38899 | 0.40 uM |
38900 | 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 |