Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:109562326-109562457 132bp AGCCGGAGAGACAGGTAAGG CCTGCCACCAGTATCATGGA
Mut= 134 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGGGAGGAGAGTAAGGATGACCAGAAGGATGCCGAGGAGGAGAGCGAGGACCAGGCTGGCCGAAGTAGCCGGAGAGACAGGTAAGGAGAGAGGGTAAAAAGTGGTGGTCAGAAAATATTCGCCCAGgttcacctcagctcctgtcggacacctaagagctttacggttttattgtgattccatgatactggtggcaggtaggagctggtaaggatttgcggggaaggctgctaagaagtggacgtaagtgcttgggttctagccctgtccaggccttcagcaccagggagatgcagtggagaggacatactgcttgttggcatggtcggaggtagagggaaatgttttctgccatggagacccctttcttcactctccctgtttcaggcattaccgatctgctcgggtagagactccgtcccaccccgggggtgtgagtgaagagttttgggaacgcagtaggcagagagagcgggatcggagggagcacggggtctatgcctcatctaaagaagaaaaggatcggaagaaggagagatcccgggatcgagactatgaccgcaagagagacagaggtaacgtgcctggcacccctcctggggtccgttagcatcgaaccagcaagcctttctcttcctgtcacaaggacttttacagtaatagtcattaattggacaacttggtttgtatagtgtgtgccccggtgGTTTTGACCCTTTGAGACTAGGATTAGGAAGAAGTTTAAGGAACGATACCTCTCTTGTAGGACAGCAACTTTGCTTGAGTGTGTGGATCTTGTTTTATTGTGAAAATTGGTGATAAAGGAAATGATGGGTGATCCATTGAAGAAGCCAGTATTTACACTA
This mutation is a 580 bp deletion beginning at Chromosome 8 position 109,561,818 bp and ending after 109,562,397 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42282 | AGC CGG AGA GAC AGG TAA GG | Common | A | |||
42283 | CCT GCC ACC AGT ATC ATG GA | Wild type Reverse | A | |||
42284 | GCA AAG TTG CTG TCC TAC AAG A | Mutant Reverse | A | |||
42285 | Fluorophore-1 | CCT CAG CTC CTG TCG GAC | Quencher-1 | WT Probe | ||
42286 | Fluorophore-2 | CCC AGG TTT TGA CCC TTT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42282 | 0.40 uM |
42283 | 0.40 uM |
42284 | 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 |