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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 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= 119 bp
Wild Type = 119 bp
Sequence
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): CTCCCAAGTGCTGGGATTAAAGGCGTGTGCTACGACCGCCCAGCGGGAAGATTTTAAATAAACTAGGTAAACCATCTAGCCCGTGTCtagtaagtgcttggtaacaaactgctgtgtaggataataaggaaatacactttcctgatcggctctaggaactttgtgaaggaagctgaggagatcagctccagccgccgcatgaacacgctgactctaaaccggcacacagaaatcctggagattttagagataccccagcttatggacacgtgtgtccgaaacagctatcatgaagaggcgctggagcttgccgcctatgtacggcgactggaaaggaagtactcctccatccccgttatccaggtagccaccttgcccaggGGCCTCAAGCTGATGATCTGATAGTTAATTCTGTGATTGTGACTACACTTACACGTAGAAACCTTTGCATAGTTCATATCTTTCTGCCAGCCAGGTAAGCTG
Mutation details
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49597 | GCC CAG CGG GAA GAT TTT A | Common | A | |||
| 49598 | CCT AGA GCC GAT CAG GAA AG | Wild type Reverse | A | |||
| 49599 | TGC AAA GGT TTC TAC GTG TAA G | Mutant Reverse | A | |||
| 51514 | Fluorophore-1 | CCG TGT CGG CCT CAA GCT | Quencher-1 | MUT Probe | ||
| 51515 | Fluorophore-2 | AGC CCG TGT CTA GTA AGT GCT TGG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49597 | 0.40 uM |
| 49598 | 0.40 uM |
| 49599 | 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.