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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = ~650 bp
Internal positive control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14463 | GAG ATG TGG CAG TTT TGC ATT | Transgene Forward | ||||
14464 | GGA CAT GCT TTG TCC GAA G | Transgene Reverse | ||||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | ||||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
Primer 1 | 0.25 |
Primer 2 | 0.25 |
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 | -- | -1.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | Go to step 2-4, 10 times | |
6 | 94.0 | -- | |
7 | 50.0 | -- | |
8 | 72.0 | -- | |
9 | -- | Go to step 6-8, 28 times | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
Primer 3 | 0.25 |
Primer 4 | 0.25 |
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 | -- | -1.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | Go to Step 2-4, 10 times | |
6 | 94.0 | -- | |
7 | 50.0 | -- | |
8 | 72.0 | -- | |
9 | -- | Go to Step 6-8, 28 times | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |