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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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= 123 bp
Wild Type = 109 bp
Sequence
Wt Sequence:GATGCCTTGCGAGGAGTGAAGCACGTGGTCCAGGTCCGTGGGAAGGAGGAGCTTGACCTTGGCCAGTGGAGCGAATGGTCCCCAGAGGTCACGGGCACTCCTTGGATAGgtacctgggtctatgtggagggaggatgggacagctttcagcttgtgagtctatcagccgtcttgcactgcatggcaaagcaggctaaatcagtcagtgtgcgctcttcatttcagttcattgagaaagggcttcattcagtccgggcggccttggaacttactttgtagtcaacatgaccttgaacttctggttctcctgcctccatctcccaagtactgggtcattctgtgtgccacagtacctagCTctatttgcccaggtctggtagctcctgtgtgtgtgtgtgtgtgtagggtcaaggtgctggctgcccatgtgtttgatggtggagctcactgggtttctccttacccactacacgtcttctgtccttctagtagg
Mutant Sequence:ACATGACCTTGAACTTCTGGTTCTCCTGCCTCCATCTCCCAAGTACTGGGTCATTCTGTGTGCCAcagtacctagcGGCCGCTCTAGAACTAGTGGATCCCCCGGGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGTCGATCGACCTCGAATAACTTCGTATAGCATACATTATACGAAGTTATT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 34709 | CTG CCT CCA TCT CCC AAG T | Common | A | |||
| 34710 | GCA CCT TGA CCC TAC ACA CA | Wild type Reverse | A | |||
| 34711 | TCC TAT TCC GAA GTT CCT ATT CTC | Mutant Reverse | A | |||
| 34712 | Fluorophore-1 | CGC TCT AGA ACT AGT GGA TCC C | Quencher-1 | MUT Probe | ||
| 34713 | Fluorophore-2 | CAG TAC CTA GCT CTA TTT GCC CA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 34709 | 0.40 uM |
| 34710 | 0.40 uM |
| 34711 | 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.