Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:22823048-22823183 136bp ATTCACAAAATGTTATGTCAGGGTTA CCCAGGGAACTGATTCATAAGT
Mut= 144 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TATTGCGGGATCCTCCGGTAGTACTATGTCTAATTTTCTGAGGAACCGCCAGACTGATTTCCAGAGTGGTTGTACAAGCTTGCAATCCCACCAACAATGGAGGAGTGTTCCTCTTTCTCCACATCCTCGCCAGCATCTGCTGTCACCTGAATTTTTGATCTTAGccattctgactggggaggaataatcacatcaatttgtaaaccaattggtaattgacttattcactttgttagattgacttttctttcaggttgcttttgatataagccattttcttttcttattcatgtttgttctttagtgtgtttactagattcaaaaataacaattgctgcttcataacaaggttaccacttagttaaattacctatatgaataaaaccagagacacattgcttcccatctttcagaacttagtcaacactagttgtagatgatctagcatattacttaaactgtcaatctttcttcctaacagatgactctgctgcagagagctctaatggcaatgagactctggggcacagctcagctgcttcagggggagcacatggcagggagcccgaggactccagcagtgatggcaaaaccgggctggagcaggaagagcagccactgaacctgagtgacagccccagctctgcacagctcacttcagagttcaggatagatgaccaaggcagtgatgggaaaaacaagtataagaatcttctaatctctgacctcaagatggaacgagaagcgagagaaaatggaagcaaggtaaggcttcactacattgctgagtgggctttatgttggtcatgaagattaagttggtaacacatgaacattactaataaatgcataaccatgcattatgtatattcacaaaatgttatgtcagggttaatagagccaagtgaaatttatctcaaagctgtgGTTATTATTTGATATAGAGAATTGTTTGATTTAATGCATTTCATTTCTATAATCTACTTATGAATCAGTTCCCTGGGAATTTTCCAAGTGCAAATGTAAAAGGTTTGGTCTCAGTTCACAATTTTGAACAATAAAAGGATCACTCATTTCTCTA
This mutation is a 763 bp deletion beginning at Chromosome 18 position 22,823,125 bp and ending after 22,823,887 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46197 | ATT CAC AAA ATG TTA TGT CAG GGT TA | Wild type Forward | A | |||
46198 | CCC AGG GAA CTG ATT CAT AAG T | Common | A | |||
46199 | TGG AGG AGT GTT CCT CTT TCT C | Mutant Forward | A | |||
46200 | Fluorophore-1 | GCC AAG TGA AAT TTA TCT CAA AGC | Quencher-1 | WT Probe | ||
46201 | Fluorophore-2 | TGT CAC CTG AAT TTT TGA TCT TAG GTT A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46197 | 0.40 uM |
46198 | 0.40 uM |
46199 | 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 |