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This assay cannot distinguish hemizygotes from homozygotes if used to genotype a transgene.
Transgene/mutant = 204 bp
Internal Positive Control/wt = 297 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
22887 | CGC CAC CAT GTA CCC ATA C | Mutant Forward | ||||
22888 | GTG GTA CCG TCT GGA GAG GA | Mutant Reverse | CHRM3 | |||
oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | ||||
oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTP KAPA | 0.20 |
oIMR9020 | 0.50 |
oIMR9021 | 0.50 |
22887 | 0.50 |
22888 | 0.50 |
10x Loading Dye | 0.50 |
Kapa 2G HS taq polym | 0.01 |
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 | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |