For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 003780
Protocol 3940: Restriction Enzyme Digest Assay - Sgsh<mps3a>
Version 2.0
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 = 105 bp
Heterozygote = 105 bp and 90 bp and 15 bp
Wild type = 90 bp and 15 bp
Heterozygote = 105 bp and 90 bp and 15 bp
Wild type = 90 bp and 15 bp
Separated by gel electrophoresis on a 3% agarose gel.**
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1419 | GGT CTG TCT TCC TCA GCG | |||||
| oIMR1420 | GAT AAG GCT GTG GCG GGA CAG GG |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| AB PCR BufferII | 1.00 X |
| MgCl2 | 1.50 mM |
| dNTP | 0.20 mM |
| oIMR1419 | 1.00 uM |
| oIMR1420 | 1.00 uM |
| DNA Loading Dye | 0.69 mM |
| Taq DNA Polymerase | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 64.0 | -- | *-0.5 C per cycle |
| 4 | 72.0 | -- | repeat steps 2-4 for 12 cycles |
| 5 | 94.0 | -- | |
| 6 | 58.0 | -- | |
| 7 | 72.0 | -- | repeat steps 5-7 for 25 cycles |
| 8 | 72.0 | -- | |
| 9 | 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 | |
| MspA II |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 37.0 | -- |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.