Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:116972090-116972187 98bp GCTTGAGGGTGACCTGCTAC TCCCCCTTTTCACTTTCTTC
Mut = 101 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCCTGGCGTAGTTGGCTTGAGGGTGACCTGCTACTTTCTCTGCAGGTCCATggctggggaaggacctcaccaactgcctgcatctggtcaaagaagaaagtgaaaagggggagggccagagaaggcaccaatggtggcaacgactgcttgttctgtatgagtctttcccaagggagtctggagccccggtccctcttggaggttgggaaactggaggcgggtgctgaggccgaggtgagcacccagaagagctggagctctgagaagaactggagcggcctctcccagggccccggcactgcttccagagagcagtctaacaaactctgcattcccaccgacgtgcatggagaaaaaaaaagcctgcaactcaaggagtttatctggtgcatggaggagtggcctatgcccgagacagttagcagcaaggccggcaggaaccccagcggaagccccgagcaagggctctccacccctgactcccttgcagccaaggctcttgtggttcttcccccgctgaaaagctctccccacaacttggacgtcctgagtaagaaaagcaggaatattttctggcagccggaagaaaaggtgctgagggtggaaaaagacgattgtatggcttgtgcagatggactgaaaggagtagacgggaaaggtgaaaagagacactttgagctggccagccacgtgaaggtcaccaacgtactgcctttcccacccactgcggcccagacccacctgctgtcagccgagtcccaaaggtgctgcctgcactggtccctcctgccccagaaaagcactgtgttcccacctaaccccagcgacatccactaccttgccaccctgcaggttttggggcaacagggaaagcaaagctgcagaactaggctcaaaaccaaggacacaaaacctcccaggaccactgccaagcatattatcacagaggccaagcagcaaaacaggccccacgtgctAGAAAGCAAAGTCTTCCCTAAACCTCTCTTGCCA
This mutation is a 926 bp deletion beginning at Chromosome 8 position 116,971,225 bp and ending after 116,972,150 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49930 | GCT TGA GGG TGA CCT GCT AC | Common | A | |||
49931 | TCC CCC TTT TCA CTT TCT TC | Wild type Reverse | A | |||
49932 | GGG GAT GAC AAC TCT GCT CA | Mutant Reverse | A | |||
49933 | Fluorophore-1 | TGG GGA AGG ACC TCA CCA AC | Quencher-1 | WT Probe | ||
49934 | Fluorophore-2 | TCT GCA GGT CCA TAG AAA GCA AAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49930 | 0.40 uM |
49931 | 0.40 uM |
49932 | 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 |