Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:109721070+109721188 119bp TGCAAACTGTATTACCTGAAGTCTG CACATAAACTGGTCACTTGTTCTGA
Mut = 117 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AATGGTAGTTATAGCTGATTTGTGCCCACCAAGCACTGATGCAAACTGTATTACCTGAAGTCTGAAAATGTTTTTTTCATTGTAGCTGCAAACTgttaaatggacagctgctcaggatgtattcttacaaaaatcagaacaagtgaccagtttatgtgttccaggtgacagtgaaggtgacgaagaggagaccacacaagatgaagtatcttcccacacatcagaggaagatggtggggtagtcaaagtggaaaaggaattagagactgcggaagcacctgtcagtagtggggacgcactagcagagcgcgaggcaagtgcacgcaggcccggccggccggcagctttccctccgccccagcctgcaccccccgaagctctcccttcctccccagcagagtgagaactactctcaggagcttctctctcaggaggatgtcttcaggttatatcagtcaggctgtcttgtggaaccagcttccaaatacattgtatcctaaggattcatgccaggaacatgtgaaagggaatctagtcctttccctgcagagtcctgtgtcctcggggcagaaagccttgaaaattgaagaaatttaatttcttttattcaaacgatattttatccttcagtctgcagtgggaacctaagactactgaaggaggtggggcagggaagagactagtgaagagtcttcatgacttgcctgtttggtggatttgcaggtcacagaaaacctaaattctgaccccttacttcaactctgccagtgtcccctctgccagctagactgtgggagtcgggagcagctgattgcacatgtgtaccaggtatggaatggggagggagctctggggctgagtgccagcagtatagaggtcagatgctgcagtggccgttgcctctgcaaaggaagctcaggttattggttctactaggactccttcctcccctcatagcatcttaggaaataatggttgcattctcatacagatagatacactgtagaattaagtatcatggtcttgtttTACTAAATTGTTTAATACAGTCAGTAGTGAAATAAACTAAACTGACTTAACAATGACGGCTTTTAAAGTGGGGTAGGGCCAACATAAATTATATGATGTGAGTCAGTAAGATGGCTCTGGTGGTAAAGCACCTGCT
This mutation is a 935 bp deletion beginning at Chromosome 8 position 109,721,125 bp and ending after 109,722,059 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50642 | TGC AAA CTG TAT TAC CTG AAG TCT G | Common | A | |||
50643 | CAC ATA AAC TGG TCA CTT GTT CTG A | Wild type Reverse | A | |||
50644 | AAG CCG TCA TTG TTA AGT CAG | Mutant Reverse | A | |||
50645 | Fluorophore-1 | TGG ACA GCT GCT CAG GAT G | Quencher-1 | WT Probe | ||
50646 | Fluorophore-2 | CTG CAA ACT TAC TAA ATT GTT TAA TAC AGT CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50642 | 0.40 uM |
50643 | 0.40 uM |
50644 | 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 |