For in-depth product & services help, ask our
Technical Information Scientists
Notes
This assay does not work well without the use of a Hotstart Taq (We are using KapaLR Taq).
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
After AccI digest
Mutant = 134 and 559 bp
Heterozygote = 134, 559, and 678 bp
Wild type = 678 bp
uncut = 678 bp
Mutant = 134 and 559 bp
Heterozygote = 134, 559, and 678 bp
Wild type = 678 bp
uncut = 678 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 11105 | CAT CTC CAG TCA GGG CTG ACC | Forward | ||||
| 11106 | GCT GCC TGT GGC CGG ACT CG | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| KAPA LR HotStart Buf Buffer does not contain MgCl2. | 1.00 |
| MgCl2 | 2.00 mM |
| dNTP | 0.20 mM |
| 11105 | 1.00 |
| 11106 | 1.00 |
| KAPA LR HotStart Taq Initial Denature 3 Min This is a two enzyme system. One with proofreeading the other without. HotStart is provided by antibody. | 0.03 |
| DNA | 0.00 |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 66.0 | -- | |
| 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 |
|---|---|
| ddH2O | |
| Rest Enzyme Buffer | 1.00 |
| Acc1 REST ENZYME: STOCK is 10000U/ml | 2.50 |
| DNA |
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.
Strains Using This Protocol
This is the only strain that uses this protocol.