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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= 112 bp
Wild Type = 109 bp
>chr9:65042263+65042371 109bp GCCATGTCTGGTGAGAACAG AAAGATCCAAAAGGGGCTTC
Sequence
Wt Sequence: AACTTTGGCAGTCTTGTTTTTCTTACTCAGGTTTTTTGTTTTGTTTCATTGTTTTTTGTTTTTTGTTTTTTGTTTTTTTTTTTTTGTTTTGTTTTGTTTTGAGACAAGGTTTCATTTAGCCTAGGCTGGCTTCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTCcacggcatgaagcatgtgtataccctacttaataacatcattttggttcctgtggaatggaaaatattataatatgtagaagacattatgtatttacgactgttgtaataaggacaatattacaaaataataatgtattttttgtttttgtttttttccttatagccatgtctggtgagaacagagaaaatacactattttattctgaaatccctaaaaccatcaacagagcagcagtcttaatgctttcctggaagccccttttggatctttttcaggtaggaacaagtattcttagtcctatgatttggtgttatttcttcatttatttcagggcaaatgtttttCTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCCAATAATTTTGGTGTTTCATTTGTTTGAAATGTTTTGGTTTGTGTTTGTGAATGTTTTGCTGGCATGTCTGTCTGTGCACCATACACATGCCTGGTGTGCATAGGCATTAGAA
Mutant Sequence:TCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTccTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46332 | GCC ATG TCT GGT GAG AAC AG | Wild type Forward | A | |||
| 46333 | AAA GAT CCA AAA GGG GCT TC | Wild type Reverse | A | |||
| 48449 | Fluorophore-1 | ATT CTG AAA TCC CTA AAA CCA TCA | Quencher-1 | WT Probe | ||
| 49159 | Fluorophore-2 | ACC ATG CCT AGC TTA AAA CAT TGT | Quencher-2 | MUT Probe | ||
| 49160 | TGT GGG GTT ATA GGT GTT TGC | Mutant Forward | A | |||
| 49161 | TCT ACT TAA AAT ATA AAA CCC TAG GAA | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46332 | 0.40 uM |
| 46333 | 0.40 uM |
| 49160 | 0.40 uM |
| 49161 | 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.