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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 =t/t
Heterozygote = c/t
Wild type =c/c
122 bp
Sequence
GAGGCCCTAATGTCGGAACTGAAGGTCCTGAGCTACCTGGGCAATCACATGAATATTGTGAACCTGCTTGGCGCATGCAc/tGGTGGGAGgtgagtctcgagttgccatctctctggttatcaa
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 17087 | TTG ATA ACC AGA GAG ATG GCA AC | Reverse | A | |||
| 17089 | Fluorophore-1 | CGC ATG CAT GGT GGG AG | Quencher-1 | MUT Probe | ||
| 23173 | GAG GCC CTA ATG TCG GAA CT | Forward | A | |||
| 23174 | Fluorophore-2 | CGC ATG CAC GGT GGG AG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 17087 | 0.40 uM |
| 23173 | 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.