Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:126438013+126438116 104bp CGGGACTTCAGCATTCTATCA ATGGGTTGAATAGCCGACCT
Mut = 119 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CAGATACACACGGGACTTCAGCATTCTATCATTAGACCTACCCAAcccaactgtctacctctggacaatgcaaccctacctcagaagctaaaggaggtcggctattcaacccatatggttgggaagtggcacttgggattttacagaaaagactgcatgcccaccaagagaggatttgataccttttttggctctcttttgggaagtggtgattattatacacactacaaatgtgacagtcctggggtgtgtggctatgacttgtacgaaaacgacaatgcagcttgggactatgacaatggcatctattcaacacagatgtacacgcagagagtgcagcaaatcttagccacccatgatcccacaaaacctctatttttatatgtggcttaccaagctgtccactcaccacttcaagcccctggcaggtactttgaacactacagatccattatcaacataaacaggaggaggtacgcagccatgctctcctgcttggatgaagcaatccacaatgtgaccctggccctaaagaggtatggtttctataacaatagcattatcatttactcctcagataatggtgggcagcccacagcaggaggaagtaactggccactcagaggcagcaaaggaacatattgggaaggaggcatccgggcagttggctttgtgcatagcccacttctaaaaaacaagggaacggtatgtaaggaacttgtgcacatcaccgattggtacccaaccttgatttcactggctgaaggacagattgatgaagacattcagctagatggatacgatatctgggagaccataagcgaaggtcttcgttcaccccgagtggatattttgcacaacattgaccccatttacaccaaggcgaaaaatggctcctgggcagcaggctatgggatctggaacactgcaatccagtcggccatccgggtacaacactggaaactgctcacaggcaatcctggctacagtgactgggtcccccctcaggctttcagcaatctgggcccaaaccggtggcacaatgaaaggattaccttgtcaactggcaagagtatctggcttttcaacatcacggctgatccgtatgagagggtggacctatccagcaggtatccaggcatcgtgaagaagctgctgcggagactgtcacaattcaacaagactgccgtgcctgtcaggtaccccccgaaggatcccagaagcaaccctaggctcaacggaggagtctggggtccgtggtataaagaggaaaacaagaaaaagaagtcaaacaaaaccaaggctaagaaaatgcaaaagaaaaaaagtaaggcaaggatgaggaagcagttggcagctcactcctcaataaaatgccacccaagtgttgccactggctaaaTGCAAAACTTTCTGCCTTGGTTAAACTTAGACTGCCTGGTTTCTAGGTAATTCAAAGAAATTGGCTCAGAAATACCTCGGCCAACCGCCTCGGGCTTG
This mutation is a 1357 bp deletion beginning at Chromosome 3 position 126,438,048 bp and ending after 126,439,404 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52402 | CGG GAC TTC AGC ATT CTA TCA | Common | A | |||
52403 | ATG GGT TGA ATA GCC GAC CT | Wild type Reverse | A | |||
52404 | TTG GCC GAG GTA TTT CTG AG | Mutant Reverse | A | |||
52405 | Fluorophore-1 | ACC TCT GGA CAA TGC AAC CC | Quencher-1 | WT Probe | ||
52406 | Fluorophore-2 | CCA ATG CAA AAC TTT CTG CCT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52402 | 0.40 uM |
52403 | 0.40 uM |
52404 | 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 |