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Mutant = ~180 bp
Heterozygote = 207 bp and ~180 bp
Wild type = 207 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
27200 | GGG CCA AAC ATA TTC AAA CC | Wild type Forward | A | |||
27658 | CTA TGT ACC AAA TTC ATG TAG AGT TGC | Common | A | |||
oIMR2088 | AGA CTG CCT TGG GAA AAG CG | Mutant Forward | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 0.24 X |
MgCl2 | 0.48 mM |
dNTP KAPA | 0.05 mM |
27200 | 0.12 U |
27658 | 0.12 U |
oIMR2088 | 0.12 U |
Kapa 2G HS taq polym | 0.00 U/ul |
EvaGreen | 0.24 X |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |