Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:10879170+10879260 91bp CATTCCCCAGTCATTCCAGT GAGTGACAGCACAAGGATGC
Mut= 86 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCTTGCTCACAAAGAGTTCATCGTGTACAGAGACCATCAACAGCAGCTTCCTCCACCCCTCAGACTGTTTGTTTGTAATAAGCACTGAGTTTCCACACTGATGTTGTATCATCTGATCCCAGCCTCTGTCATTTCCTCATTCCCCAGTCATTCCAGTCAGGTCAGCCCCAAAGCCCTGCAGgttgtgacatagctccatacctgaaaagcatccttgtgctgtcactctagcagtgggacttaagcttgtggtacatctgggtttttttgtttggttggtttggtttggtttttcaagacaggatttctctgtataatcctagctgtcctggaactcactttgtagaccgtggcctcgaactcagaaatccgcctgcctctgcctcccaagtgctgggactaaaggcgtgcacgccacggctggcttggtgcatctttccttaaggtccagatgaaggttcattattgtgtgtgcatgctttcttttcagatcaacagaaacatttaaaaaagaagatgatcttgacagtcttattaatgaaatattcgaggagcctgactttgacagaaaatcttttgtaagtcactaaatgtacagtaattatttaagatgcttagactcgtcttaaagttaagtcacagtagccatctgggtatgagaattctgaggagtacaggtaaagaaaGGATTCCACTTTTTAAAATGTGGAGGGATAAAATGTTAGAAACTCTGTGTGTATTTCTGTTAACTCCTCAGTGAGAGTAAGAATTTGAAAAGCTGTTATTAGAGAAGTCAGCTCACAGACCACATTGGTCACTTAGAGTTCGTGGTAGTGTCTAAGAACGTGCTCTAAAAG
This mutation is a 505 bp deletion beginning at Chromosome 4 position 10,879,214 bp and ending after 10,879,718 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46964 | CAT TCC CCA GTC ATT CCA GT | Common | A | |||
46965 | GAG TGA CAG CAC AAG GAT GC | Wild type Reverse | A | |||
46966 | TTT CTA ACA TTT TAT CCC TCC ACA | Mutant Reverse | A | |||
46967 | Fluorophore-1 | TTG TGA CAT AGC TCC ATA CCT GAA | Quencher-1 | WT Probe | ||
46968 | Fluorophore-2 | CCC TGC AGG GAT TCC ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46964 | 0.40 uM |
46965 | 0.40 uM |
46966 | 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 |