Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:99219394-99219500 107bp GTTCTGTCCCGACATCCTGT GAAAGTCAAAGGGGCTGGT
Mut = 156 bp
Wt = 107 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TAAGTACAGTAAAAATATTGATCATAGTTAATACATATGTGTTTGGTAGTATGACAAATTAACTACCTTGAATTGACTTGAACAGTTAGAGACTTAGAGTTCATTTAATTCTCTACGAGTGTGAGCAGTGATTAGtggtgcccttagcttaatcagtttcaaatatttatgagcaactgacagtggtgagtaagatccagtgggataaggagggcatggggtaagccagctgttggcagttttgaaacaggctgattgctacaaaggagtttgatactgtcttctttgctttttcttgtatttaaacgtttccatattagagtgtttttaaatgactatatacttaggcactaatattgaattttcaactattgactttttaaggtctgggaccaagtaaaggcttccaaccctgacctaaagttgtgggagattggcaagattattggtggcatgtggcgagatctcactgatgaagagaagcaagaatatttaaacgaatacgaagcagaaaaggtaggaggactgccttggcttattatttataaaagaaaccaagcgtgaccagatggctgagccacactgcagttttgtccaccaagcttcactcatctgagtggcacattctttgttctgtcccgacatcctgttttacctctccctctgatgttcagagggacagtgcaggtggcctgtggagtagaatggacattataaaaccagcccctttgactttcagatagagtacaatgagtctatgaaggcctaccataattcccctgcgtaccttgcctatattaatgcaaaaagtcgtgcggaagctgcattagaggaagaaagtcgacagagacagtctcgcatggagaaaggagaaccttacatgagcattcagcctgctgaggatccagacggtaaggtggaaaacggggctttgtatatgtattctctctgaattagaaaagaaatttagttcaaggcatttttattcttttaaatggaaaagcataattttaagtctttttgttgtagtaagattttaagtagtaagatttttgcgtgtgactttctcatggaaaaaatactccttacagggtGGAAAATTATCAAAGAAAAAAAGCTGTATTTATTACTTATCAAAGTCTTTATACTCAGAATAGAGGATTTGTTTGATTTTCGAGACAGGGCCTCACTGTGTTGCCCA
This mutation is a 955 bp deletion beginning at Chromosome 11 position 99,219,039 bp and ending after 99,219,993 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50050 | GTT CTG TCC CGA CAT CCT GT | Wild type Forward | A | |||
50051 | GAA AGT CAA AGG GGC TGG T | Wild type Reverse | A | |||
50053 | GTC TCG AAA ATC AAA CAA ATC C | Mutant Reverse | A | |||
50054 | Fluorophore-1 | AGT GCA GGT GGC CTG TGG | Quencher-1 | WT Probe | ||
51669 | CCT TGA ATT GAC TTG AAC AGT TAG | Mutant Forward | A | |||
51670 | Fluorophore-2 | AGT TCA TTT AAT TCT CTA CGA GTG TGA GCA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50050 | 0.40 uM |
50051 | 0.40 uM |
50053 | 0.40 uM |
51669 | 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 |