Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:28368736-28368836 101bp GTGCTTCGTGCAAAGAGTGA ATGGGAGTCAGCACCACCT
Mut = 100 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCATTACCAACACCCCCTTTCCCAGGGCTGTGGTGTCACTCCCTAGGTCACGACATGCCAGCCATGCCAGGGTTATCAGGCTGAATAGTACGAAGCCGTGAGGAGGCAGAATTTCTGGGGTAGCCATCTGAGGCTCCTTCCTACCTGTCTGTTGCCCTCTggagtcttgtcatgggacaggggggcaggcaagccctttaccttcccctttgcccgcccacagtgagctcctggaggacttggaaggctgcagcagtgcagggggcatcgccgagtgcttcgtgcaaagagtgagtaggacagcttgaggctgggggacggacgctgggccgggattcatggggccatggggatctaggtggtgctgactcccatgtccccacagagcgaagattttgacatctatacattgtactgcatgaactacccaaggtgaggcaaagggagtccctgtttggcccccgggtattcccacgagccaatgtgccaaaccactaccccctaccctttaccgcccccaaccctgtgcccaggggcagccccttctagaccttgatagtcactggcctccttaactaccctgacctgatcatccctgaccctggccacctcctcccagccctgcctgctggagtgccagccaccactgatctatgctgtgccccccagctccctggccctgctccgagagctgtcagtgtccccgccagccactctgtggctacaggagcgtcaggcccagctccgtcactccctgcctctgcagagcttcttgctgaagcctgttcagcgcatcctgaagtaccatctcctgctgcaggtcagctgtgcccctgaggcctggcttgtggagagggggcatggttgggtctaacagccacccctggtaccctgactgtctgtggtctgcccctcccctttcatgttgctctgcctcctgtctcactcctcttttctctttttctcccccatccttctaccctgctggatgactgtcaggaactgggcaagcactgggcagagggcccagacagtggagggcgagagatggtggaggaagccattgtatccatgactgcagtggcctggtacatcaatgacatgaagcgcaagcaagagcatgctgcacgcctgcaggtggcccccggggtgggctggggcacctgagctgggggatgttgctcagtgtgtcccggtcactaacgcctctgttttggcttgtatatgtgtgccaccaacctgggcctgtctctatgcctgatgtggtcccagcaggaagtgcagcggaggctgggtggctggactggcccggagctcagcgcctttggagagctagtactggaaggcactttccgagggggaggcgggggaggccccaggcttcgtgggggtgagcgcctcctcttcttgttctcccggatgctgctggttgccaagcgtcgggggcctgaatacacctacaagggccacatctttgtgagtatagagtggggtctggctcagagagtcagaactaatgtatgttcccctgagttcccgaaaggggacagactggcttccaaggaccatatttagctgcgcccagtgacatttgacctttgattccagttactgggaaggctgagacaagaccaaaagttcaagactaacctgggctacagaaaaggttcaagaccagtcccgggcagcctgattaaaacaacattaacggctagaagtagGGCATGGTAGCTCACTGGGGAGGCAGGGGGGTTGAAGTAAAAGCCTGAGGCCAGTAAGACCGACAGGTGAGCTCAAGGCCAGCGTAGGTTATGTAGTGAGATCCTGTCTCAGGAGGGCACACTGATCTCCAGGTTCTAGGGTAACTAGGACTACAAAGAGAGCCAGACCCTGTCTCAAAACCCAGCCAAAAAAGCAACAGTGAGCGTAGCTGGTGGCCACAGGAACCAGTGTGTGAAGATGCCACAAACTTGTCCGCTGACCTCCACTGGCC
This mutation is a 1561 bp deletion beginning at Chromosome 7 position 28,367,390 bp and ending after 28,368,950 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49136 | GTG CTT CGT GCA AAG AGT GA | Wild type Forward | A | |||
49137 | ATG GGA GTC AGC ACC ACC T | Wild type Reverse | A | |||
49138 | GGC TCC TTC CTA CCT GTC TG | Mutant Forward | A | |||
49139 | CTC ACC TGT CGG TCT TAC TGG | Mutant Reverse | A | |||
49140 | Fluorophore-1 | TGA GGC TGG GGG ACG G | Quencher-1 | WT Probe | ||
49141 | Fluorophore-2 | CCT CTG GCA TGG TAG CTC ACT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49136 | 0.40 uM |
49137 | 0.40 uM |
49138 | 0.40 uM |
49139 | 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 |