Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 136 bp
Wild Type = 144 bp
>chr7:109331313-109331456 144bp CTGTGTTCCTCACCATCTCTG CTGCATTGTTGCTAACTAAAACGTC
Sequence (deleted region in lower case)
GGCGCGGTAGAAACTTGTTCTAACCTTCACTACATTCCTCTTTCCACCCCGCAGCTTCCAGTACTGTTGGTACAGATCTACATGTTATTTTAATGCTGCTCCATTCAGGCTTTAGGGGTTTTGTGTGCTGGCCACCAGCTCTGCCTGGTTTTCCTCCACTTTCCACCCCCAGAAATGACTGCACACACGTTCAGTCTCAACTCTTCTCGCTCAGTGAGCATTCTTTATTTAGAGGCTGCCTCTCTTCCACAGAcccatggcatgctggctgttagcatatatcacatgatattgtaactctgctgagcttcctgaggtcatcgctatgtcgcttggccctctgctcctcgtgtattcaggacagtagcttatatagagtagataagacattttcacattaataaattagaattgtgagcatacttttgatatctaacacatggaacttcataactttctcttgttatgcctttaaacctgtaatgtcagaaaatgtatctcgtgatggagctttgtgaggatggagaactcaaagcagttatggatcaaagagggcacttctcagagaacgagacaaggctgataattcaaagtcttgcatcagccatcgcatatcttcataacaagggtaagacaagaactgtgttcctcaccatctctgtaacaggaatacatcactgttgtacattggtagagcttggtaatgcAAGAGGAGAACAGGTACATTTTCCATAGTTTCCCCTTTTAATTGTATTTATAGACGTTTTAGTTAGCAACAATGCAGTCATAAAATCAGACTGTCCGTGAAAACGTCATGATCATTCTTATTTTAGAAGTTTAAAATGGGCTAATATGATCAAAATTAAAGTAGAAAAATCAAAGTGAATTTTCCTAAAATATTT
454 bp deletion beginning at Chromosome 7 negative strand position 109331843 bp and ending after 109331390 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37688 | TCT CAA CTC TTC TCG CTC AGT G | Mutant Forward | A | |||
37689 | CTG CAT TGT TGC TAA CTA AAA CGT C | Common | A | |||
37690 | CTG TGT TCC TCA CCA TCT CTG | Wild type Forward | A | |||
37691 | Fluorophore-1 | TTT AGA GGC TGC CTC TCT TCC | Quencher-1 | MUT Probe | ||
37692 | Fluorophore-2 | TGT TGT ACA TTG GTA GAG CTT GGT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37688 | 0.40 uM |
37689 | 0.40 uM |
37690 | 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 |