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Stock No: 000575
Protocol 5580: Restriction Enzyme Digest Assay - Cacna2d2<du>
Version 2.0
Notes
BspHI has 100% activity using both NEBUffers 2 and 4. Enzyme is supplied with NEBuffer 4. Cuts at 37C. 07/23/10 LMV
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 = 286 and 273 bp
Heterozygote = 286, 273 and 541 bp
Wild type = 541 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10504 | ACC TAT CAG GCA AAA GGA CG | Forward | ||||
| 10505 | AGG GAT GGT GAT TGG TTG GA | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. | 1.00 |
| dNTP | 0.20 |
| 10504 | 1.00 |
| 10505 | 1.00 |
| DNA Loading Dye | 0.69 |
| KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. | 0.03 |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | |
| 4 | 72.0 | -- | |
| 5 | -- | Go to step 2-4, 35 times | |
| 6 | 72.0 | -- | |
| 7 | 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 |
|---|---|
| ddH2O | |
| NEB buffer 4 | 1.50 |
| BspHI | 1.25 |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 37.0 | -- | |
| 2 | 4.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.