Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:80198567-80198682 116bp GATACGGAAACATTTGGCCAT GGGATTGTGGAATAAGGTCA
Mut= 107 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCCAGGACAGCCAGGGCTATACAGAGAAACCCTGTCTCGAAAAAACTTTAAACAAACAAACAAACAAAACTTAGTATGTCCCAGAATGAGAGTGAGCTGTTTGGAGCCAGGACTGATCTTTATAATCAGACTGTTGTCTCATCCCAGCTCAAAATGGCTCCACAGAAAGTCGGCTGTAGTTTGGCACagcagagggtggacaagacacatggtgcttctggaggctaggcagtgaaaaatcgtatttcattggttcttcctttcctgtctcataggtccatccgaagccaggagttggcattaaaaatggtgagcatcccttcttgaacggctgcttgagaggtgacacacatccacaggactccagtctgaactccgtctccacacagagaaccttggagccaaggagccggaggaggagcaggagtcggaggttcaaagtacattcgctggagcttccacaacccagtggctcatgggaaaactctactgaccctttgtggagaaggcttggctggtccaccctggccgacagctccgcgtcaggcatgcagcagcgccgactgtctatccccatagaacccaagatgggaaggaacgttcttagagaagatggctttgagaagctcgaggcggagacaaatgttgacactggcttcaccatcatcaattccgaacccaggcgatcttttccgagcagatttcccagggattttcagagcagccaaccaccacgctgcttgtcaggctcttccttagaggtaactgtttccccaaaccaaggagtctttaaggatagactccaccctctgtcacttcctagtcaaagggactttggtgcaaagaaggtcatctcaaaatgttcccatcatctgcaggcccagggtgaagagaaagaagaattctttatatagcataaaacaattgtggttttttttttcctaagacacgagaatgttatagaaaccaagcgttgtattagttgcctttgtagttgaaatgagaaatatcctacaaaagcaagcaaaggtttaagagtcagccccattcatggctgggaagtcacagaagcaagagtgtgactggttacgtttcctctacagtcaggtagcagagggggatggatgctggcgatctgcctgcctttttaaaagctcaatctgggactccagctcatgggaagatattacccatttttagggttggtcttccttcctcagttaaatctttctggaaacacccttctagatacacccagagatgggtttccatggtgtgtctgaatccagttgacagtgaagatcggatctacgaacatttagtttcttgctcctttgagagagatctaggctctcgatgcccagtattagaagtccacggggatctgacagtgtgctggccagttggaagggagagaaagaaaacccagggtctgagcttctcagagacacagctcagatgcatgtgttggctcagcacctagcagggccttggcactgtgtggaaatgaatcagatgtgctatgcaaatctgattagtgtgtgtaatatggttttaatttttccttgaggttgcatagcttatccccttggacagtaggaactatttattctttagtgcgttttaatttttgtgtggcctcctttggtaagtaggccctgtttacgatacaccctcttgtggttttgcctttaaagttctttgcatatgaaacgagtgaagaatgcaaagtgttttaaaattataaataccaccccctcccctgtttaattagtaacaatactcgaagccagaaagctaggttcaaaatgagtgctagactcagagatatttctactcttttgtctctcatttgttcatgtcctacttgaaagagatcatcaggtcttgttaaaacaaaaacaaacaaacaaacaaacaaacacaaaaaaacaaacaaaacaaaaacaaacaaacaaacaaaaccaaggaagacagggatgattagaaaattctgtatctccactgagcatgccgaaaggttgtgttggctgagttgctagaccaggacttgagttctagctgtattttgggggctcctggggcttgtatgttcacgtaataaacatttttttttgcatgcagtaaatattcatggatatgatacggaaacatttggccatcaaattcaaaaagtattcatggctttgcaactgcaaccctccacTGAACCAACTCTTGAAAAAGGTGTTTAAGTTTGACCTTATTCCACAATCCCAGCCCACATTTGCCATGTAAAGATGTACTAAACTCTTAGTTTTCTCATCGTTTTCATGAAACACCCCAGCAAAAGCAAATTACGGTAGGGAGGGATTATTTTGGCTGGTAGTTCCAGGTTAGAGTCTAT
This mutation is a 2019 bp deletion beginning at Chromosome 1 position 80,198,618 bp and ending after 80,200,636 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44540 | GAT ACG GAA ACA TTT GGC CAT | Wild type Forward | A | |||
44541 | GGG ATT GTG GAA TAA GGT CA | Common | A | |||
44542 | TGT TGT CTC ATC CCA GCT CA | Mutant Forward | A | |||
44543 | Fluorophore-1 | AAA AAG TAT TCA TGG CTT TGC AAC | Quencher-1 | WT Probe | ||
44544 | Fluorophore-2 | CGG CTG TAG TTT GGC ACT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44540 | 0.40 uM |
44541 | 0.40 uM |
44542 | 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 |