Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:6062321+6062431 111bp TGTGACAGCTGAGGAGATGG CCAGTTCAGCCAGGGAGTTC
Mut = 120 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AAAGAAGTACTGGAGACACGAAAGTTACCGAGAAGTTTCGGAAATGTGCGGAAAGGAGGCCACGCCCGCCAACCACCCCCACACATAAAGGCGGAAATAAccaaagaagcgccggaagtggcggagctgatacgggacaggcggcaccagtttccatggagcccgctgggctctgcggcttctgcccggccggggaagcgctgcctgcgcgctacacctgcccgcgctgcaatgctccctactgctcgctgcgctgctaccgggcgcacggcgcctgcgccgaagacttttaccgcgaccaagtgctgcgagagctccgtggccgaagcgcttcgcccagccgtctggcgggcgcgttacgccggctgcgcgaacaacgcgaggccgaggatgagcccgaggaagcaggcctcgggcccggggcgcggccgggcggcctttctggactttgggaacgtttaacgccggctgagaaggcggcgttcgagcggctgctgagccgtggcgaagccggacgccttctgcctccgtggcggccctggtggtggggccgcggcaccggcccacgtttgctggaggagctggatcatgccgcgaacagagatcttgcggagccggagcccgcccctgcgaggaccgcgctgcaatctggggatgatgccgctgccgcagagccatttgctgaagactcctgtgccgccagaccgctagcattgcctgctcgcatcccagcactggccagtctgagccgcagcccggcatcgccgctggtgcgcttccagctgcccaacgtgctgttcgcctacgctcatactctcgccctgtatcatggaggggacgacgacgcgctactctctgacttctgtgccaccctgctcgatgtttctggggccctgggagcccagcaagtctttggctctacagaggaagccctgcaggccgcagcccacgtactggaagcgggcgagcacccacctgggcccctgggtacgcggggtgctatgcaagaagttgcccgaatcctgctgggcgagggtccagtcaatcagaaaggctacactctgacagcactggggcacctagctcagaccctgggacgagcccggaagcaggctgtgattggtggagagcgagatcgactttaccgggctcggaagaagtgccagttcctgctggcttggaccaacgaaaacgaggctgccctcacacccctggcgctagactgtgccagagcccaccgagcccatgctgtgacagctgaggagatggcaacgcttactggagagttagaacggctttggggaggcccagtgccacccactccaaggactctcattgaggaactccctggctgaactgggtatccatgtcattaataaagatgtgtctggtctgcccgaatgtcagcagtgccttttccgtaaagatagtgccctgtcctaggcttctcttgaagttactgtgtagttgaggatgaccctggaactttgatctgtcacccacctggacagtgagaggttacaggggttaaccaccttgctaatgtacacagtgctgaatagccgaggcgtcctgtgtgctacacaagcactctataactgaactacatcacatcccccaggcctgaggtgcccagttgtctctcctcgtcagacttttggacCCACAAACCACTTCTAAGCTTTCCTTAGTCAACTAGTCACCCCCAAGCCTCAAGTCACTGGCCCCAGGATAGAAGAAAAACACAGGAAAGGTTGAAAACATTTATTCAACAGGGAAAAATACTTTCCTTCTGGAAGCAGTGGCTTCCTCTTGCTTTCAACCT
This mutation is a 1563 bp deletion beginning at Chromosome 19 position 6,061,172 bp and ending after 6,062,734 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51563 | TGT GAC AGC TGA GGA GAT GG | Wild type Forward | A | |||
51564 | CCA GTT CAG CCA GGG AGT TC | Wild type Reverse | A | |||
51565 | AAC CAC CCC CAC ACA TAA AG | Mutant Forward | A | |||
51566 | CTT TCC TGT GTT TTT CTT CTA TCC | Mutant Reverse | A | |||
51567 | Fluorophore-1 | CTT TGG GGA GGC CCA GT | Quencher-1 | WT Probe | ||
51568 | Fluorophore-2 | AAC CAC AAA CCA CTT CTA AGC TTT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51563 | 0.40 uM |
51564 | 0.40 uM |
51565 | 0.40 uM |
51566 | 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 |