Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:120790560-120790661 102bp GCTTCAAATGTCTTCTGTGAGG TCTTTTCTCAGCTGTTTTCACTGG
Mut= 109 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGATCCACCTACCTCTGCCTCCCTGCTGGGATTAAAGGTATACATGGCCACTGCCTGGCTCAATACTTGATTTTCTAAATTGGAACTTTTTGATAGTCTGTGATGTAATCTAGGAAAATTTTGCTTCAAATGTCTTCTGTGAGGATGTAATAAAGCCTATCAGCACagcacagaccttaacactgaatgaaacaattaaaccagtgaaaacagctgagaaaagataagtaactagcctggtgagaaagtatgactttattatttctaatgtgctttgtgtctgtgtgttttcagttgtgaaaatgtccacagaaggagggtttggcggtaccagcagcagtgatgcccagcaaagcctccagtccttctggcccagagtcatggaagaaatccgaaacttaacagtggtaaggaaaaacaaggagcttctaaagcagatcagtagaaacaagactcggGCACTTTTATTTTCAAGCATATTCAGGCGTAGTGTATTCCTGACCTGAATGTTGGGAAATAAAAATAGAATTTAACTTGTAGGAGGCAGTACCAAGGTGTGTTTGTGACAGTGGCCATCTGTTTAGATGTTCTGCTTGGAGGCAGGCTCATGTGGAACTGTGCTTTAGCCTATAGA
This mutation is a 292 bp deletion beginning at Chromosome 4 position 120,790,326 bp and ending after 120,790,617 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44147 | GCT TCA AAT GTC TTC TGT GAG G | Common | A | |||
44148 | TCT TTT CTC AGC TGT TTT CAC TGG | Wild type Reverse | A | |||
44149 | TTT CCC AAC ATT CAG GTC AG | Mutant Reverse | A | |||
44150 | Fluorophore-1 | AGC ACA GAC CTT AAC ACT GAA TGA | Quencher-1 | WT Probe | ||
44151 | Fluorophore-2 | CAG CAC TAT CAG TGC ACT TTT ATT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44147 | 0.40 uM |
44148 | 0.40 uM |
44149 | 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 |