For in-depth product & services help, ask our
Technical Information Scientists
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 = A/A
Heterozygote = G/A
Wild type = G/G
Sequence
tacccttgagcccggccgcttggctttcactcccaccctctgcccacccggctgctccctcctc
tccctcctcctcgggtgcagGACGGGCCTCTTCACACCTGACCTC(g/a)CTTTTGAAGCCACAG
TGAAAAAGCAGGTGCAGAAGCTCAAAGAGCCCAGTATCAAGTGTGTGGACATGGTAGTCAGT
GAACTCACGTCCACCATCAGAAAGTGTAGTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 27326 | AGG ACG GGC CTC TTC ACA | Forward | A | |||
| 27327 | GGC TCT TTG AGC TTC TGC AC | Reverse | A | |||
| 27328 | Fluorophore-1 | CTG ACC TCG CTT TTG AAG CC | Quencher-1 | WT Probe | ||
| 27329 | Fluorophore-2 | CTG ACC TCA CTT TTG AAG CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 27326 | 0.40 uM |
| 27327 | 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.