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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 = ~500 bp
Heterozygte = ~500 bp and 350 bp
Wild type = 350 bp
Heterozygte = ~500 bp and 350 bp
Wild type = 350 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
Sequence
Wt Sequence:
CTATGAGATACAGCTGCCAGCGCCGGGCACGCCTCATCACCCAGGGCGAGAACTGCATAAAGGCCTTCATAGACTGCTGCAACCACATCACCAAGCTGCGTGAACAACACAGAAGAGACCACGTGCTGGGCCTGGCCAGGAgtgagtggtggcttgtagggtgacactcaggtggggatgggggaacacaactgcccagtgctgggcctggccaggagtgaatggtggcttgtagggtgacactcgggtggggtggggggacacaactgcccagtgctggacctggccaggaatgagtggtggcttgtagggtgacactcgggtgggggtgggggagcacaactgcccagtgatgggtggctgcacctgtttgctagaagagtggcccaaagactgtgcccccaaccttgtcccctgggaggctagaaggaggaggatggaggccagctgggcagtgt
Mutant Sequence:
GCCAGCAGCCCGCCGCCGTCGCTCAGTACAGTTGATGGAAAGAAGGATGGACAAAGGTTAGGAATCCTCTACTGACCCACATCTCCATGCATCACCCACACTCCTCCCATAGGAGGAGGAGGGCGTTGCCTTCCTTGTCTCCCTGAAGTCTGGTCTGATGATGTCCTGATGGCTAAGGCTGACATCAGGTCTGGGCCTCCCACAGCTGGTCAGTACACTGACAAGGGTCTTCGGAAGTGTTGTGAGGATGGTATGCGGGATATCCCTATGAGATACAGCTGCCAGCGCCGGGCACGCCTCATCACCCAGGGCGAGAACTGCATAAAGgaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR1325 | ATC TTG AGT GCA CCA AGC C | Common | A | |||
| oIMR1326 | GGT TGC AGC AGT CTA TGA AGG | Wild type | A | |||
| oIMR7415 | GCC AGA GGC CAC TTG TGT AG | Mutant | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR1325 | 0.50 uM |
| oIMR1326 | 0.50 uM |
| oIMR7415 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.