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This assay cannot distinguish hemi from hom
Tg= 145 bp
IPC = 132 bp
Tg Sequence:
tgttggcaaaggggttttcttaatcagactgttttttcggtccgaggaaaaggaggtaggagattgtgatggagaaagggggtctgtaaaacgtcaggcgCCGCACAAAGGCTTTGCCACGTAATTACAGGCTCCTATTAAGTGGAGATCCCCCGGGCTGCAGGAATTCGCCGCCACCATGCCCAAGAAGAAGAGGAAGGtgtccaatttactgaccgtacaccaaaatttgc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33394 | AGG GGG TCT GTA AAA CGT CA | Transgene Forward | A | Prrx1 | ||
35249 | TGA ATC TGG CTT CCT CAA CTA TTC T | Internal Positive Control Forward | A | |||
48147 | GTA CGG TCA GTA AAT TGG ACA CC | Transgene Reverse | A | Cre | ||
48148 | Fluorophore-1 | CGC ACA AAG GCT TTG CCA C | Quencher-1 | Tg Probe | ||
oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33394 | 0.40 uM |
35249 | 0.40 uM |
48147 | 0.40 uM |
oIMR3580 | 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 |