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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
>chr13:59474519-59474648 130bp AATGAAAGGAACACTGGAGT CTGCTTAACTTACTTTCCATTGA
Mutant = T
Wild type = C
Sequence
SEQ
tagctgttcattttaattgagcttttgtgtgcgctttagGAACTCGCCCTTATATTTTCTTGTCTGCTCGGGTCCATCCTGGAGAAACCAATGCAAGCTGGGTAATGAAAGGAACACTGGAGTACCTCATGAGCAATAGCCCGACTGCCCAGAGCCTA(c/t)GGGAGTCTTACATTTTTAAAATTGTCCCCATGCTAAATCCAGATGGTGTCATCAATGGAAAgtaagttaagcagtggctgccggagtgccataggttgggatagttggctgcatttaatttgttcatctaatagtgttcaagttaattcaaataatgggtttgatgattttttttctagg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68700 | Fluorophore-1 | CCA GAG CCT ATG GGA GTC TTA | Quencher-1 | MUT Probe | ||
| 69001 | Fluorophore-2 | AGA GCC TAC GGG AGT CTT | Quencher-2 | WT Probe | ||
| 69179 | AAT GAA AGG AAC ACT GGA GT | Forward | A | |||
| 69180 | CTG CTT AAC TTA CTT TCC ATT GA | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 69179 | 0.40 uM |
| 69180 | 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.