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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 =G/G
Heterozygote = A/G
Wild type = A/A
chr18:12196478-12196560 83bp AGGCCTCAGGGGAAAGAA ACTTACCCTTTGCCGCACT
Sequence
TGGCTCTCCTTTTTTTGTCAGTGATGGACCCAACCTGTGTCCGCTGCAGACCTCTGA
CTCCAGAGGGTAAACAGAGGCCTCAGGGGAAAGAATTCATGAAATTCCTGCCCATG
TTCCTTTCTG(a/g)TAACCCCAACCCCAAGTGCGGCAAAGGGTAAGTGCTGGCCAGG
GAGCGCTGAGCTAAGCAGTCACTGCTGCTTCGACCTGAACCAGAAGCAGAGTTTG
GATTGAGCAGGCTTGTACGGAAGGGTCCTCTGCCTGCCCTGCCCTAGTCCTGTAGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59889 | AGG CCT CAG GGG AAA GAA | Forward | A | |||
| 59890 | ACT TAC CCT TTG CCG CAC T | Reverse | A | |||
| 61617 | Fluorophore-1 | TCC TTT CTG ATA ACC CCA A | Quencher-1 | WT Probe | ||
| 61618 | Fluorophore-2 | TCC TTT CTG GTA ACC CCA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59889 | 0.40 uM |
| 59890 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.