Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:18715442+18715541 100bp GCATTTTCAGCACAGTTTGA TGGGAACTGCCTGGGATATG
Mut= 107 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTAATATGATTTTTAGGTATATATCAGAGCATAGCTGCTATTCAGTGTAGTTCACTGAGGTCAGGAAAGTCTGCCATACTGAGAGGCTGAGGGGAAATAACAGAAAAGAGGTGGTCAGTCAACAGAGTTGGTTAATAGGCTAACATTCCAGAGTTCCCCTGAGTCAAACAGTACCTTTTAGTATCAAGCAGATGTAGAGAATCCAATTATTATATGAATTTGCAGAAAGTCCCATGTTTATATCTGATTTTATATAATTTCCAAATACATGCTTAAAAGTAAAGCATTTTCAGCACAGTTTGAAACTGACCTTGCTTCTAGGTTAGTTACTAACcagcggcaatgggagcgataaagactcaacatatcccaggcagttcccaccgtctgcgttccgaggcggttgtgctttccttcaatgccagttttgtgtcactttcaggctattttagattctaggtttggtctctttttccctgtagtgtgtctacaagaaggcagctgcgtttgtgctgcgagcggttggtaaacactctcctcagctggcgcaggccacagttgactgtggtgccctggactcactggtgatctgcttggaagattttgaccctggagtcaaggaggctgcagcctgggcacttgcatatattgcaaggcataatgcaggtactggggtgtgcttgaagatagactcaagtttaactttactagataagaacagttttgtttattatccattgatggagttttaaaagttgctggacattatgggagagctgtcttgaaggatagccacactgatttggatcacataattacaaaacacttctacatttgagttctagcagaagggaatctgaaggagacGGTGGTATGGTAGAGTGGGATTGGCATCCAGTAGCAAAGGTACATGCCTATAATCCCAGTATTCAAGAAGCAGACTCAGAAAGCTTAAAGTGAGCTCTGCAAGCCTGAGTTACACAGCCAGACACTGTCACAAAACCAAAACAACTAAGTCAGATGAAGAAAGTTCAGCCGACCAGGTAAATATATTCTTACTAACTTGTCAGAAGACTATTACTGGTTTCTCATGCTCTGAGCTCCTTGTCATTCACGACAGGACATTTCAAGCCAATCAAGGAGCCAGATCACATGGATGAATGGTGCGGTAAGATAATATACAGTTGTAGAAAATGTTTTATTACCCTGTGTTAGCAGGAAGGAGGAGGAAGAAAATAAGGGCAACACTTTTCTCAAAACTGAAATCCTGGTGTGGTCAACAAGACAAGACGGGGCTCTCCTGAGCGAAGACT
This mutation is a 533 bp deletion beginning at Chromosome 2 position 18,715,493 bp and ending after 18,716,025 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48272 | GCA TTT TCA GCA CAG TTT GA | Common | A | |||
48273 | TGG GAA CTG CCT GGG ATA TG | Wild type Reverse | A | |||
48274 | GGA TTA TAG GCA TGT ACC TTT GC | Mutant Reverse | A | |||
48275 | Fluorophore-1 | AAT GGG AGC GAT AAA GAC TCA A | Quencher-1 | WT Probe | ||
48276 | Fluorophore-2 | ACT AAC GGT GGT ATG GTA GAG TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48272 | 0.40 uM |
48273 | 0.40 uM |
48274 | 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 |