Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:46245331+46245434 104bp ATGTCTTCGTGGGAAAGACG GGCTCTCTGTAGGCCACACT
Mut = 113 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGGGAGGAGGGTGGCAAGTGTCCCCTCTGCTCCTTCCCATTCCCCATGGGGACACCGGATTCCTGGGGCTAGGTAAGAATTTCTGGGGAGATCCCAGGATCCAGGGGCCAAAGTGGGGCAAGAACACAGAAAACTGGAAAATGATGGGTCAAGGCTTTCTCATGACGCAAGAAGGAGATGGTTTGAGGTCTGGCGAGCACgaagtcaggcttcacgtaccaaggaggggtggcaagcttggctggacctgccttttcttgagagacctctgctgggagccaggcttcatcttcggcatgtttaggtgtagaggaaagaggacgtggagccagatagcccagtgcacagaactaattgttctctgttcctaaaaggaggctgaagccttagccgcatcggccatgtcttcgtgggaaagacggctccatcgcgccaagtgtgcaccgtcgtgtaagttgcgccctctctgtggctatgcgtaggagagtgtggcctacagagagcctgccagggtaccacttgtgagccccttccggtgcctggagtctcttcctaccttccccagcttgatttgcatagcctcaccgtatcctggtcacatccttgtgtcccggctcctgctcacattgtctgattcgtgagtttgctgcctcacaagatggctgactcctcctccgacctcatactctttctggtcttgcttcctacttccagtgggatGATCCTTCAGTGTGCTAATTCCTGTGGTTAATTCTCTCTTTACCTCCCTTAGATCTAAGGCCTTCTCATCCCCCTTTTTCTGTCTGTGACCTAGTCCCATGTCCTGTGTCTGTGCGGGCCCCCATCAGAGTCCCATGTCTGTCCTGTCCGTGAC
This mutation is a 520 bp deletion beginning at Chromosome 7 position 46,245,130 bp and ending after 46,245,649 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48921 | ATG TCT TCG TGG GAA AGA CG | Wild type Forward | A | |||
48922 | GGC TCT CTG TAG GCC ACA CT | Wild type Reverse | A | |||
48923 | GGA GAT GGT TTG AGG TCT GG | Mutant Forward | A | |||
48924 | CAG ACA GAA AAA GGG GGA TG | Mutant Reverse | A | |||
48925 | Fluorophore-1 | TCG TGT AAG TTG CGC CCT | Quencher-1 | WT Probe | ||
48926 | Fluorophore-2 | CAC GAT CCT TCA GTG TGC TAA TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48921 | 0.40 uM |
48922 | 0.40 uM |
48923 | 0.40 uM |
48924 | 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 |