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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:5305360+5305506 147bp TGCAGCCAGCTTTATTATTAGAC GGCAAAAGAAAGGGTGACAG
Mutant= 108 bp
Wild Type = 147 bp
Wt Sequence: TGCAGCCAGCTTTATTATTAGACTTAACTGTTCCTTTAAATCATTTCCTTTTGTCTTGTACCATATATAGATCTCTAGATCTCCAATTCTAGAGTCTTCTGAACTCTTGCAGTCCACCATCtgAATTCTGTCACCCTTTCTTTTGCC
Mutant Sequence: ACTTGTGTAGCGCCAAGTGCCAGCGGGGCTGCTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAgaattctgtcaccctttcttttgcc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46312 | TGC AGC CAG CTT TAT TAT TAG AC | Wild type Forward | A | |||
46313 | GGC AAA AGA AAG GGT GAC AG | Common | A | |||
46315 | Fluorophore-1 | CTA GAG TCT TCT GAA CTC TTG CAG TC | Quencher-1 | WT Probe | ||
46316 | Fluorophore-2 | CTG CTA AAG CGC ATG CTC C | Quencher-2 | MUT Probe | ||
oIMR0950 | ACT TGT GTA GCG CCA AGT GC | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46312 | 0.40 uM |
46313 | 0.40 uM |
oIMR0950 | 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 |