Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:115820986+115821119 134bp CCTGATAAAGCCGATTGTCC AGCCTTCTCCCACACCTCA
Mut = 120 bp
Wt = 134 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAGTGAAGGTGGGTGACTAGGCCCTTGTGGCTGTTCCCAGGTACCCTTCCTTCTGCTATGGCTCTGCCTCTGACCTTTGCCTCATCCTCTTGGCCAAGCTCTCCCAAATCCCATCTACGgtggcggcagcctgagtgaacggacccgcctcctgttctcacccacagctctgtcgtgtcaccttacgagcggcagcttaatttggatggttatgcccagtgcctggaggatgggtctggcaagaggaagaggagttccagctgtcggtaatccacctgctgctgccacccctgggagcacatgggcttaagaaggggcctgaggacacagccgagggtatacctcagttccagtggaatgtacgtgtcttacggacagtaacaagggacaaatgtgctcttttttctcccgtgtctctaaggtctgttaataagaagcccaaggtcctgcagaactccctgccgcccgtcagcctggcagcctccagctcacctgcctgtgaccagagtagccaatacccagaggagaagtctcaggactcaagccccaggcagggctcagagctccccttgcagttcctggggtcctcagagccttgctctgatctggccagggaagacgtggggtgtgaccgagagagtcacaaaatagagaatggagctaagggaacccctaagctacgctggaactttgagcagatctcattccccaacatggcctctgatagtcgccacactttcctgcctgctccagccccagagctgctcccagccaatgtcattgggcgagggacagatgctgagtcctggtgccaaaagctgaaccagcggagggagaagctctcccgtagagaccgggaacagcaagcagtggtccagcagttccgggaggatgttaacgcagatcccgaggtgcggggctgctccagctggcaggagtacaaggagctgctgcagaggcgacagacgcagaagagccagccccggcctccacacctatggggccagtccgttacccccttgctcgaccctgataaagccgattgtccaggtatccctttaccctgagctatacccctataggctgttgggagcagagagagacttctgaaagtggatgcttagacgAGGGCTGGCTAAGGCCTGAGGTGTGGGAGAAGGCTGTGCAGTGGGGTGGGACCTATATAGGCTGTTCCGTGGGTGGTGGTGGCAGCCACTGGGGACAGTTTCCTGATGAGTCATTGACCAGTGATTCCCGTGGGGGATGAAAG
This mutation is a 1008 bp deletion beginning at Chromosome 11 position 115,820,077 bp and ending after 115,821,084 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50627 | AGC CTT CTC CCA CAC CTC A | Common | A | |||
50628 | CCT GAT AAA GCC GAT TGT CC | Wild type Forward | A | |||
50629 | TCC CAG GTA CCC TTC CTT CT | Mutant Forward | A | |||
50630 | Fluorophore-1 | CCC TGA GCT ATA CCC CTA TAG GCT G | Quencher-1 | WT Probe | ||
50631 | Fluorophore-2 | TCC CAA ATC CCA TCT ACG AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50627 | 0.40 uM |
50628 | 0.40 uM |
50629 | 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 |