Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:121978231+121978363 133bp TTTTGGTTTTCAGGGTTTGG GCAATCTCACCTTTCCTGAG
Mut= 115 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTTCCTGTGTGCGCCATGTACATGGTAGCTTGTGACAGGACTGGCCCTTGGGAGAGCAAAGCCATCCTGTTGAGCTTACCGAGCACATGGCCTGCTGCTGACTTCCAGCTTGCTAGTTGGAAACTGCTCCTCTTGTGGGCCACTAGGTGGCACACTTGATATTTAGAAGGTCCCTGGAAGGGGTCATTGACAGATTTCAGTGAGGTATAGCTGTGTCCAGCCATCTTTTTTTGGTTTTCAGGGTTTGGATAGTAGTCTTATCTTTCAGCATTAGAAAAATTTTGTAGCTTTACTGTataaatatttggatgagcatagtgacaagcttattactccatggcctcaggaaaggtgagattgcaagggcttattagacacagagggctgaatccctgtgcacggcccagggctcctcactgaatatgttggctcggcccagggctcactcagctgaaatacactgggtggacttaggagccagctagggtcctaaggacttgctgctcacagtggaagacatcctctctgaagcttttctgttttcatttccagtctttcctgtactctattaaccagacgatatgtttgcggttggatagcattgaggccaagctgcaagctctcgaggccacttgcaaatctctggaagagaagctagacctggtcaccaataaacagcacagtcccatccaggtccccatggtggcaggttccccacttggcgccacccagacctgcaacaaagtgcgatggtaagtccagaagaccgaggctccgcaggccaccctgggaaagcgctccgtgccagcccaggcgccgctgttactgatctgccctcgccccggATATCTGCCCTGGTGCTTCCATGGGGCTGGTTTCACTTAGACCGAAACCAAACACAGGCTCTGCTGGAACGGCCTGAGTTGCTCCAACTTTGTTTCTTGGCTTAAGCAGCTGGACTCATCTTTCCTTGCACCTGGCCATGAACTTGGCCCATGTATGTTGCTTCTTTTCTCTTTCTATCTCTGGCTGACTATTGCCTATGTTTTCTGCGGGGGTAATATTTATAATTTTTACTTGATCATAGAAAACTTTCCGTTTAGTCTGAGACTTGCCTTGTGCCTAATTT
This mutation is a 549 bp deletion beginning at Chromosome 8 position 121,978,299 bp and ending after 121,978,847 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48288 | TTT TGG TTT TCA GGG TTT GG | Common | A | |||
48289 | GCA ATC TCA CCT TTC CTG AG | Wild type Reverse | A | |||
48290 | TTT CGG TCT AAG TGA AAC CAG | Mutant Reverse | A | |||
48291 | Fluorophore-1 | TTG GAT GAG CAT AGT GAC AAG C | Quencher-1 | WT Probe | ||
48292 | Fluorophore-2 | AGC TTT ACT GTA TAT CTG CCC TGG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48288 | 0.40 uM |
48289 | 0.40 uM |
48290 | 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 |