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Tg tm=89
IPC tm=83.5
Tg= ~300bp
IPC=200bp
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7303 | TCT GAG TGG CAA AGG ACC TTA GG | Transgene Forward | A | |||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control | A | |||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control | A | |||
| oIMR9296 | CGC TGA ACT TGT GGC CGT TTA CG | Transgene Reverse | A |
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7303 | 0.50 uM |
| oIMR8744 | 0.50 uM |
| oIMR8745 | 0.50 uM |
| oIMR9296 | 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 |
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