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Stock No: 007228
Protocol 41485: End Point Analysis Assay - Pbk1-rs31442355 (near D1Mit15) Alt 1
Version 2.0
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 =C/C (c3H)
Heterozygote = T/C
Wild type = T/T (b6)
Sequence
AAAGGGATGGAGGGAACAAGAGGGAATGGGACCTTGGATCTAAGGTGTAGCTTCCATGTGAATTGATATGTCTAAGACACGGAGGAAGTGTGTGTTTAACCCACATGTGCCCAGAGGGAAAGACAGGATACATCCATGTTCTTTGTTTTGCTTCCTCGTTGACCCACCAGCCTGGCTGATTGAGGGCCTGCGGCACGTACACCT(t/g)GTGCTTCCACTTCCATGCTGAAGGCATCTCTGATAATGGCATCTGGCCCGTATGCAGATCTAGCTCAACTTGGTGCTGACATCAGCCAGTTTGTGGCACCTCAGCGTATTTCTGGGTTGCAAATGAGAGTTTCTTAGTGCCAGGCTCGCTTGGGCTGTCAAATCAGGTGATGAATTCTCTGTATCAACAGTGCGCTGCTAAGGTAAGCGCAGGGGAAGAGCGGGAAGGAAAGGAGATGGGACAGGAACTGCTTATAGCCCACCCACCAGCCTAAAGGCCCCGCAAAAGCCAGGGAGAGGCAGCTGCCTGTCGGCCCCTGTTCCGGTTCAAGAAAAGTAGCAGAGAAGAAAGAAATTTTCCAGCCTGCCGTTGCCTGAGGCTTGCTCTGGGAGCTCATTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCAGACCACCATGCTCAGTCTTTAGGTATACTTCTAGCAAAATCTGTCCCAGAGTGGCCTATAGAATAGAGAGTCACTTTTGTTT
Red is nearby SNP used to genotype
Blue is Mit
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 58094 | CAG ATG CCA TTA TCA GAG ATG C | Reverse | A | |||
| 58095 | TTC CTC GTT GAC CCA CCA | Forward | A | |||
| 58467 | Fluorophore-1 | CGT ACA CCT TGT GCT TCC A | Quencher-1 | WT Probe | ||
| 58468 | Fluorophore-2 | ACG TAC ACC TCG TGC TTC CAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 58094 | 0.40 uM |
| 58095 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.