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Stock No: 000632
Protocol 551: Restriction Enzyme Digest Assay - Lep<ob>
Version 1.1
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 = 55 bp and 100 bp
Heterozygote = 55 bp, 100 bp and 155 bp
Wild type = 155 bp
Heterozygote = 55 bp, 100 bp and 155 bp
Wild type = 155 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 |
|---|---|---|---|---|---|---|
| oIMR1151 | TGT CCA AGA TGG ACC AGA CTC | Common | ||||
| oIMR1152 | ACT GGT CTG AGG CAG GGA GCA | Common |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 0.89 X |
| MgCl2 | 1.78 mM |
| dNTP | 0.18 mM |
| oIMR1151 | 0.89 uM |
| oIMR1152 | 0.89 uM |
| DNA Loading Dye | 0.62 mM |
| Taq DNA Polymerase | 0.02 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 62.0 | -- | |
| 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 |
| Dde I |