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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:110777584-110777691 108bp CAGGCAGGTTGTATCCCTGA GTAGAAGCCGCAGGCAAGTA
Mutant= 146 bp
Wild Type = 108 bp
Wt Sequence: CAGGCAGGTTGTATCCCTGAAAACCATGGCACAGATATGAGGAAGATGTCCTTCCAGCGGTGTGCTCGAACAGACaAGgtatgtgtcctacttgcctgcggcttctac
Mutant Sequence: GCCCAATTCCGATCATATTCAATAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGAGGGGATCCACTAGTTCTAGAGCGGCCGACAGCTGAaggtatgtgtcctacttgcctgcggcttctac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
24716 | GCC CAA TTC CGA TCA TAT TC | Mutant Forward | A | |||
46207 | CAG GCA GGT TGT ATC CCT GA | Wild type Forward | A | |||
46208 | GTA GAA GCC GCA GGC AAG TA | Common | A | |||
46209 | Fluorophore-1 | AGG AAG ATG TCC TTC CAG C | Quencher-1 | WT Probe | ||
46210 | Fluorophore-2 | TGT ATG CTA TAC GAA GTT ATT AGG TCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
24716 | 0.50 uM |
46207 | 0.50 uM |
46208 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |