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Mutant = ~250 bp
Heterozygote = ~250 bp and 333 bp
Wild type = 333 bp
>chr12:112852719-112853051 333bp CTGGGCCTACCAGCCAAC ACACGCAGAAATGGTGACAG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50272 | AGT CTG AGG TGC GAG AAA CC | Mutant Reverse | A | Mut R | ||
50989 | CTG GGC CTA CCA GCC AAC | Wild type Forward | A | Wt F | ||
50990 | ACA CGC AGA AAT GGT GAC AG | Wild type Reverse | A | Wt R | ||
oIMR2088 | AGA CTG CCT TGG GAA AAG CG | Mutant Forward | A | Mut F |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
50272 | 0.50 uM |
50989 | 0.50 uM |
50990 | 0.50 uM |
oIMR2088 | 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 | 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 |