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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 110 bp
Wild Type = 110 bp
Wt Sequence:
GTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGTCAATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT
Mutant Sequence:
GCTCCATGGGTGGCACTTGGCGGGGTGCTTGCTTACAGACCGGCCTCTCTGTGTCTCCACAGGTTTACAAGTCGTCCTGAAGctgATCATGAAGGCGATGATCCCTCTGCTGCAGATCGGCCTCCTCCTGTTTTTTGCAATCCTTATTTTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
67170 | GGG TGC TTG CTT ACA GAC C | Forward | A | |||
67172 | AAC AGG AGG AGG CCG ATC T | Reverse | A | |||
67173 | Fluorophore-1 | CGT CCT GAA GTC AAT CAT GAA G | Quencher-1 | WT Probe | ||
67175 | Fluorophore-2 | CGT CCT GAA GCT GAT CAT GAA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
67170 | 0.50 uM |
67172 | 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 |