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Size on ABI 3730.
JR6471 Transgene = ~596 bp
JR4938 Transgene = ~500 bp
JR2810 Transgene = ~ 724 bp
JR6494 Transgene = ~ 574
C57BL/6J = no band
Human = ~ 266 bp and 280 bp
There are 168 bases upstream and 53 bases downstream of the repeat for a total 221 bases to subtract to get the repeat length.
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR6533 | GGC TGA GGA AGC TGA GGA G | |||||
| TmoIMR1594 | Fluorophore | CCG CTC AGG TTC TGC TTT TA |
| Component | Final Concentration |
|---|---|
| ddH2O | 0.00 |
| Kapa2G Buffer M (wit | 1.00 |
| MgCl2 | 1.20 |
| dNTPS-kapa | 0.20 |
| TmoIMR1594 | 0.28 |
| oIMR6533 | 0.28 |
| Betaine | 0.50 |
| DMSO | |
| KAPA2G Fast HotStart | 0.01 |
| DNA (crude prep dilu |
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | |
| 4 | 68.0 | -- | repeat steps 2-4 for 10 cycles, decreasing annealing temp 1.5 degrees C for each subsequent cycle |
| 5 | 94.0 | -- | |
| 6 | 50.0 | -- | hold |
| 7 | 72.0 | -- | repeat steps 5-7 for 28 cycles total |
| 8 | 72.0 | -- | |
| 9 | 10.0 | -- |
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