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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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
Sequence
Large deletion: Mutant sequence with junction in lowercase
TTTTAGTGGGAAATCAGATCTTATTAGCCACCAAAGGACTCATACCGGTGAAAGGCCCTACAAATGCAATAAATGTGAGAAAAGTTACCGACACCGGTCTGCCTTcaCGTGTGTCTTGACAAGATGTCATGTAACCTTAAAAATATATTTACATATCAGAATTCAATAAGTGAAAGACTCCTAG
This mutation is a 512 bp deletion of Chr13:23,356,351-23,356,862 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 80561 | CCT ACA AAT GCA ATA AAT GTG AGA | Common | A | |||
| 80562 | CCA AAG CAC TTC TCA CAG G | Wild type Reverse | A | |||
| 80563 | AAG GTT ACA TGA CAT CTT GT | Mutant Reverse | A | |||
| 80564 | Fluorophore-1 | AGA GTT CAT ACT GGG GAG AA | Quencher-1 | WT Probe | ||
| 80565 | Fluorophore-2 | TCT GCC TTC ACG TGT GTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 80561 | 0.40 uM |
| 80562 | 0.40 uM |
| 80563 | 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.