Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:104847907+104848043 137bp CCCTGGTAACAATGAGATGGA TGGATCTGTCCTGTATGTGTGT
Mut = 159 bp
Wt = 137 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTGAACCCTGGTAACAATGAGATGGAAATTTCACAATCTGCTGGCAGCTGATTACAGAAAActaacccagaggcatctctccaccaggtcaggactcaccagaggccaaccccatcagaaacacacatacaggacagatccaaggcagccttatccacgtgaaggacactaaagctggtgtccacaccttcctgggaatcccctttgccaaacctcctgtaggaccactgcgatttgcacctcctgaggcccctgaaccatggagtggtgtgagagatggaactgcacatccggccatgtaagttttggacccagagggtttcaggcactgggggagaatgtactctgagctctgtgctatgctgaagtgtttcctccacctcagtgacacagcagttttctctctttgtggatgagtgccccatgggtttttctgatgatgtgctaatgttgattagaacaactgggccaggctcctaggtcagagcttggcactcatagggaataacatggatatactatggagaaaaACAAAGATAAAATTGTTCACACATTCCAATTAACCAAAGTGTTCTCTGCCACAATGGGTGCATCTCTAGGCATGAGTAAATGTCAC
This mutation is a 469 bp deletion beginning at Chromosome 8 position 104,847,963 bp and ending after 104,848,431 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51569 | CCC TGG TAA CAA TGA GAT GGA | Common | A | |||
51570 | TGG ATC TGT CCT GTA TGT GTG T | Wild type Reverse | A | |||
51571 | TGC TTG CTG CCT AAT ATG TGA | Mutant Reverse | A | |||
51572 | Fluorophore-1 | CCA GAG GCA TCT CTC CAC CA | Quencher-1 | WT Probe | ||
51573 | Fluorophore-2 | CTC TGC CAC AAT GGG TGC AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51569 | 0.40 uM |
51570 | 0.40 uM |
51571 | 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 |