Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:80795805-80795900 96bp CCTGAACCCTGGTTGGACAC GGGTTACCTACAACTCCCACT
Mut= 96 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTAGGGTCTAACCTAATGACCTTCTATTAGCCACTTCCATTCCCAATAACCCAACTTCCCAGTGAGTCCCCTATTTGTGGGTTCAGGGGTCAGGATTCTGGAGGACAAAGCCAGCATGAGACAGGCAGAAGATGACTTGGTGTTAGAATAAGAGGTGTAGTTTGAAATCCTGgtgagaggaagctgggacttgtggccaacaggatgtagaccaactggacactgaccagaggtggctcttatcctttatccctttccaggcttcgtcgaagacctcagcaggaaatatcactttgaatgctgtagccaagagcagtgtcaggactggatggaagctctgcagcgggccaggtggggctttagggactgatgggagctgggtggaaacatgggggaatgtgaacagtgagcccaatcctttcctccagctacgaatacatgcgacagagcctcatcttctacaggaatgagatcaggaagatgactggcaaggtatgtggggcggtcagggatgggacagctttccctggccttgctcacagccaccgtgccctccacaggaccccctggagcagtttggcatctccgaggaggccaggttccagctgaatagcctgcctaaggatgggcggactacttgtattggctcccagttaaatgtgttggactgaccctctctgggcccctggctaccctgggaagtttttttttttttaatataggttttaatacatctgttcaaaaggtccagaggaggtgggcagggtctgtggtcctcgttctccatgccatctccctgccagcaccccttgagtgtccacatccagggaccgagcctggcacagctcccctatccattgccagatgaatgtataatgctagatgaagctgtgaaccccccccccccctcctgctgatagagcagggcatgggtgctggcctcagtgctcagcctgctctttaataaaggtacctcttttctgagccatgtcggaatgagtggagaagaggagggtgggacacaggctgtgggcacttgggaggcagggggaagaggagggtgggacacaggcagttccagagcatctgggacagagaacaaaacaggcaagctgggctggtggcacacacagtataggtgtaggcaggacttggggggacaagcctctggctcgcagtgtgcctgaaccctggttggacaccctcactgtggagatgtggatccatccttggGTTTTGGGGAGTGGTACTCATATAAGTGGGAGTTGTAGGTAACCCTGATGGGGAAAGGTGTGTTCTGCCCAAGTTGTAAGGGACACCTAAATCCTGGCTTTCTAAACAGGCTGTGTGCACTTGGGAGGCAGCGGGAAGAGGAGGGTAGGACACAGGCAGTTC
This mutation is a 1061 bp deletion beginning at Chromosome 10 position 80,795,850 bp and ending after 80,796,910 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44015 | CCT GAA CCC TGG TTG GAC AC | Wild type Forward | A | |||
44016 | GGG TTA CCT ACA ACT CCC ACT | Common | A | |||
44017 | CAG GCA GAA GAT GAC TTG GTG | Mutant Forward | A | |||
44018 | Fluorophore-1 | CTG TGG AGA TGT GGA TCC ATC | Quencher-1 | WT Probe | ||
44020 | Fluorophore-2 | TTT GAA ATC CTG GTT TTG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44015 | 0.40 uM |
44016 | 0.40 uM |
44017 | 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 |