Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:44536065-44536156 92bp AATGTGACCCACGTCCTTTC GGATTAAAGTCCAAGGCTCTG
Mut = 132 bp
Wt = 157 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGTCAGTGCTCTTAATCGATGAGCCATCTCTCCAGCCTGGAGTATTTTTTTTTTCTTTCCTAAAGAAAGGGCAAGTCTGGGCAGGGGAGACTTCCCTTCTGGGTCTGGAGCAAGAAGAAAATAGACAAGCAGATACAGGCTTGGGCTGGGGAAGCCTGCATGATAGGAGAagctggcgaagatgtctgtcctctgcataatgacatgtgcctgcctcggtgctgagactgattctttgcctttgtaggtttcacaagctaatatggggaagctttggcagtgggcttctggaaaactccggggttattgctggtggaggagacaacggcacactgactctatacaatgtgacccacgtcctttcttcagggaaggagcccctgattgcccagaaacagaagcacactggagctgtcagagccttggactttaatcctttccaggtactgtgtcttagctgaccagacctggtcaaggcatgacgacgcatgtctgaatggaagcattctcttgtggccttcaggctgtgcctctgaactgtaatgggagaggccctgggtctaccaaatgcaagcctcctctttctccttttctgaggtgggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttgggggtaggtctttagaaatatcttttattcacacagggcaatctcctggcctcaggagccagtgactctgagatcttcatttgggatttgaaccacctgagtgtcccaatgacacctggacccaagtcacaggtgaggggctccctcctccctctgacctggggctgcctcttagggtgtacagccaggttttcccaggtaaagtttggaattgtggagccagccgagtaacagagatttccagctttaccactggagggcaataaaaccccacagatggacaggtcacctagtagtcaggagAGAGGGCAGAGCAGGTGGTTCACGGTGGTCAGTGCTGTTCAAAGAATTCCTGGAAAGAAGTGAAGGGCAAGGGGGGACAGAAGACACCAAGACAGGCAAGGAGTGATATGTATCATAGT
This mutation is a 697 bp deletion beginning at Chromosome 19 position 44,535,584 bp and ending after 44,536,280 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49662 | AAT GTG ACC CAC GTC CTT TC | Wild type Forward | A | |||
49666 | Fluorophore-1 | AGG GAA GGA GCC CCT GAT TG | Quencher-1 | WT Probe | ||
51671 | GGA GCA AGA AGA AAA TAG ACA AGC A | Mutant Forward | A | |||
51672 | Fluorophore-2 | ACA GGC TTG GGC TGG GGA A | Quencher-2 | MUT Probe | ||
52321 | TCC ATT CAG ACA TGC GTC GT | Wild type Reverse | A | |||
52322 | ACT GAC CAC CGT GAA CCA C | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49662 | 0.40 uM |
51671 | 0.40 uM |
52321 | 0.40 uM |
52322 | 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 |