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Stock No: 003923
Protocol 452: Restriction Enzyme Digest Assay - Cpe<fat>
Version 3.1
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
Mutant = 64 bp and 26 bp
Heterozygote = 90 bp, 64 bp and 26 bp
Wild type = 90 bp
Heterozygote = 90 bp, 64 bp and 26 bp
Wild type = 90 bp
Separate digested products on a 3.0% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0703 | CAG CTT GCC CCC GAG ACC AAG G | Common | ||||
| oIMR0704 | AAG GTC TCC TCC GTG CAG ATT GGC CG | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 0.92 X |
| MgCl2 | 1.27 mM |
| dNTP | 0.17 mM |
| oIMR0703 | 0.51 uM |
| oIMR0704 | 0.51 uM |
| DNA Loading Dye | 0.64 mM |
| Taq DNA Polymerase | 0.02 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | 72.0 | -- | repeat steps 2-4 for 35 cycles |
| 5 | 72.0 | -- | |
| 6 | 10.0 | -- | hold** |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Reaction B
| Component | Final Concentration |
|---|---|
| AB PCR BufferII | X |
| ddH2O | |
| BSA | mg/ml |
| HPa II | U/ul |