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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= 104 bp
Wild Type = 101 bp
>chr5:117203488+117203588 101bp ACACACATACGCATGCACAG TCTAGTTGTCTGGGAATAGATAGCA
Sequence
Sequence(deletion in upercase) ttagtgcctcggttggttggttgtgtgcttggttgattggttagtcagttggtcgattgggtacttgggtaggcatatggctggttggttggttggttggttggttggttggttggttggggtaggttggattgtgattggttgcttggttgattgattggctgtttggttggttttgtagctatttggttggtttggtgtggttAACTTGAGGAACTTCTCATTAACTTTTATTATTCCGATCTTCCACTCTGTTCCAGTTGGTGATGGAGTACTGCCTAGGTTCAGCCTCTGACTTGTTAGAAGGTAAGCATGACATCATAGCCTCTTCTGTTTTCCTTCTGTCACGTTCTCCATGGGGCTGGAGAGACAGCTCAGCGGCAAGAACACTCTTCTCTCTCAAGGACCCAGTTGGGCTCCCAGGCAAATGCCTGTAACTCCAGCTCTGGGGACCTGGCATCCTCTTCTGGCCTCCACAGGCACCTGCACCCAATGTGCACACACATACGCATGCACAGGCAGGCCACATgtacaggcacggacactaaaataaaatcttaaaaacaaaaaagaatgctatctattcccagacaactagacatagtgcagtttgagcctctggttgaaaggcagtttaagggtcactgggtatactgtatagacataatctctacactatagctgggtgatctcagctgcatggctgtcccgggcgggcactagccaaaaaccatcaaagccatgtataaatgtcactgcctgtgcctttccagtatcacagtgggcacggatgtgagaggggaggtgctgtcaactcactagtatctacccatgtgcagagtcaacgacggacctgagctctcccaccactgctgctgtgtgtgcccagcagctagctgtgtttaaaaattctattta
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40576 | GGT TTT GTA GCT ATT TGG TTG GT | Mutant Forward | A | |||
| 40577 | TCT AGT TGT CTG GGA ATA GAT AGC A | Common | A | |||
| 40578 | ACA CAC ATA CGC ATG CAC AG | Wild type Forward | A | |||
| 40579 | Fluorophore-1 | CAG GCC ACA TGT ACA GGC ACG | Quencher-1 | WT Probe | ||
| 40580 | Fluorophore-2 | TGG TGT GGT TGT ACA GGC ACG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40576 | 0.40 uM |
| 40577 | 0.40 uM |
| 40578 | 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.