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Stock No: 024496
Protocol 26682: Standard PCR Assay - Id3<tm2.1Zhu>alternate3
Version 1.2
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 = 294 bp
Heterozygote = 155 bp and 294 bp
Wild type = 155 bp
Sequence
Mutant Sequence:
CCAGTGAGTCTGGAGGTCAGACGAGCAGCAAATTGGGGAACAAGGCGGCACTAATTCCTTAC
AAGTTTCCCTTGAAAATTCTTGAGAGAGAGAG(aggagagagatcccccgggctgcaggaattcgatatca
agctggccgctctagaactagtggatccggaacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcg
acctgcaggaattcgagctcgccc)GATCTTTAGTTGTTCAAGGGATTTATGACCTCAGAGCTGTGGG
TTCGAACCAGTATTGGGGGGAGGGGACCTCAGAGAACTTGGGCG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 20027 | ATT CCT GAC GCC AGT GAG TC | Forward | A | |||
| 20028 | CCC ACA GCT CTG AGG TCA T | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 20027 | 0.50 uM |
| 20028 | 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.