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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 = ~230 bp
Heterozygote = ~230 bp and 151 bp
Wild type = 151 bp
>chr9:120051874-120052024 151bp AGCTCACGACTGCCTTCTTC ACGCCCAGACTAATGGTGAC
Sequence
SEQ
tgtaagcgttaatgacctgcagccaagctagcttggctggacgtaaactcctcttcagacctaataacttcgtatagcatacattatacgaagttatattaagggttattgaatatgatcggaattcctcgagtctagatgcggccgcaattcgagctCGGTACCTTGCCATCGTCCTGGCCGCCAACTCCATGAACAACCGGACAGTGCAGCACGGTGTCACCATTAGTCTGGGCGTCTGGGCGGCGGCCATCTTAGTGGCGTCACCCCAGTTCATGTTCACAAAGAGAAAGGACAACG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 20669 | AGC TCA CGA CTG CCT TCT TC | Wild type Forward | A | |||
| 21058 | ACG CCC AGA CTA ATG GTG AC | Common | A | |||
| 21059 | GTT AAT GAC CTG CAG CCA AG | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 20669 | 0.50 uM |
| 21058 | 0.50 uM |
| 21059 | 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.