Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:94867548-94867702 155bp ACACTCACCTTGGCCATTTG CAACAATGCTGCCCTGAAGT
Mut= 146 bp
Wt= 155 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGATAGGAAGCATTCCACAAATGTTCCTTCCATTACACAGAGCATCATAGAACAGCCCGATAGAGATGAACATCAACACTCACCTTGGCCATTTGGTTAGGGCTGCCCCTCTGTTGGCTGCTATCCTGTAGCTACCCTACCATCCtaaggttttctctaagctaggatccagggaagggtgtagtattgctccctcaggaggctggggtaacttcagggcagcattgttggggatcacggtccagcaacaatccgatcattagcttcttgacccacagatttaccggaggtgctggctggtgttccggaaatcttccagcaaggggccccagcggctggagaagtatcctgatgagaagtccgtgtgcctccgaggctgccccaaggttaggggcctgggtcctagatcccccaacctgtgagcaaagatgacttgctgagtcctgaggtctgagagtaccttcctggggaacccagctaaggaggtaaggtgaagtgccccggcctggctaaatctcttgggatatctttccaggtgactgagattagcaacgtcaagtgtgtcacacggctccccaaggagaccaagaggcaggcggtggccatcatattcacagacgactccgctcgcaccttcacttgtgactcaggtaaggccccaggactgcaggctccatctgctctcctcgggctggtcagtcagtggtctaggcagtgagtaccagagcaaggccaacccttgcagaaagagtgggagagctgtgcccttaGTGGAGTAGGTCGCAGACAGAGGTGGGCAAGGCAGGCAGCAAGGCCTTCCTGCATGGTGCAATGAGTCAACCCAGGGCAGATTAGGTGGGCTGCTGGGGCCTCTGAGCTGGCAGAACTATGAAGTGCTATTTTTTTCTAGAGCTGGAGGCAGAAGAGTGGTA
This mutation is a 625 bp deletion beginning at Chromosome 8 position 94,867,008 bp and ending after 94,867,632 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45585 | ACA CTC ACC TTG GCC ATT TG | Common | A | |||
45586 | CAA CAA TGC TGC CCT GAA GT | Wild type Reverse | A | |||
45587 | CCT GGG TTG ACT CAT TGC AC | Mutant Reverse | A | |||
45588 | Fluorophore-1 | AGC TAG GAT CCA GGG AAG GG | Quencher-1 | WT Probe | ||
45589 | Fluorophore-2 | CCA TCC GTG GAG TAG GTC G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45585 | 0.40 uM |
45586 | 0.40 uM |
45587 | 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 |