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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 = 127 bp
Heterozygote = 127 bp and 310 bp
Wild type = 310 bp
Sequence
MUT SEQ:
AGTAGCTACTGAAAGGCTCTTCATTGTCTCTTCTGCTCCAGGAACACCGGTCTAGGAAGCAGAAGGTAGGATTAGAAATGGGGCAGTATTCTCCTGGTACTTCGTGGTGGGAGAAACCCTGGGCTGGCAGAACACTCTAAGGTCTAGGTCTTGGATCAGTTTCTTCCTTCTTTCTCACACCAGTGAGCTCAGTGGGGCTCACATGTACACGCTCTGTTTACTATGAACCTCTCCTAGACAGGTGCTGTAGGATGCTATGTGCCCAGatcgataacttcgtataatgtatgctatacgaagttattcgaggtcgacgaagttcctatactttctagagaataggaacttcggccgccaccgcggtggAGCTCGAGGACGGCACTACTACACTTGGTTTACATAAGTAAGGCCACCGGTGTTAAGAGCTGGTCATCTTTGCCCAAGATTCAACTATGAGT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 24572 | CCT CTC CTA GAC AGG TGC TG | Common | A | |||
| 24573 | TGA TTT GGT GGA TCC TTT GC | Wild type Reverse | A | |||
| 24574 | CGG CCG AAG TTC CTA TTC TC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 24572 | 0.50 uM |
| 24573 | 0.50 uM |
| 24574 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.