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Stock No: 008604
Protocol 28920: Standard PCR Assay - Smn1<tm5(Smn1/SMN2)Mrph>
Version 3.2
Notes
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Wt Tm- 84 area 8
Het WT TM- 84 area 5
Het MUT TM 80 area 3
Mut TM 80 area 8
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR8508 | TAC CCA GAT GCA GTG CTC TTG TAG | Mutant Forward | A | |||
| oIMR8509 | CCT TAT GGC ATA GAC ACC AAC TTC T | Mutant Reverse | A | |||
| oIMR9111 | GTC CCT GGT CGA CAA GAA CAG | Wild type Forward | A | |||
| oIMR9112 | ACG CTC TGC TGC TGA CTT AGG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR8508 | 0.50 uM |
| oIMR8509 | 0.50 uM |
| oIMR9111 | 0.50 uM |
| oIMR9112 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| 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 |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.