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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
>chr12:69959047+69959172 126bp GGTGGTGATAAACCATTTTTGG GTAACGCCGGCTGAACTCTT
Mut= 116 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
tcatagtaactgtgctctgactgacaacacagttctggctgcagtgtctcgaggtggtggaagtgtagcccaccccttagtccatcactcttaggaagcattctggtgtactccaagaaaaggcatggacacagttgttaatttactccatattacataaacagaagcctctctttctgacagGTTTGTGGTGGTGATAAACCATTTTTGGCCCCTAACGACCTGCAAAGCAAGCACCTGCAGCTAAAAGAAGAATCTGTGAAGTTATTCCGAGGCGTGAAGAAGATGGGCGGGGAAGAGTTCAGCCGGCGTTACCTGCAGCAGCTGGAGAGTGAAATAGATGAACTCTACATCCAGTATATTAAGCACAATGACAGCAAAAATATCTTCCATGCTGCTCGAACCCCAGCCACACTGTTCGTTGTCATCTTTATCACATACGTGATTGCTGGTGTGACTGGATTCATTGGTTTGGACATCATAGCTAGCCTGTGCAACATGATCATGGGACTGACGCTTATCACCCTCTGCACCTGGGCATATATCCGCTACTCTGGCGAATATCGAGAGCTGGGTGCTGTAATCGACCAGGTAGCTGCAGCTTTGTGGGACCAGgtgagagtaccttcaggtttgcacatgctgtgtaattagtgttgcattcactattagaaatatacaggtgaacatgacttgggcccattctttaaaactttacagtataccaagatttgagcaactaactaagtcctacacaactgctttctagttaacattttagtatttaaaacatacatacattaacagcatttttactctctgattctaaaatattaccgtgtgagaagttaatagtgttagtagtgtctgacagtcatttagctgtcgtgaccttgagttaaacttaacttagcccttgactagcactggcttggtgaggggcatatgtgaaccacaatcatttggttatatgtcattcccttt
Mutant Sequence:
CTGTTCATAGTAACTGTGCTCTGACTGACAACACAGTTCTGGCTGCAGTGTCTCGTCCATGTCAAAAAGAGAAAAATTTAAAATGATTTTAAGCCACCTATAAAATACCAGTCCCATTTATAGTCATGCCTCGTGGATAGGGGGTGGAAAGATGTGGGCTGACAAAACATATATGCAGGCTCCTGATTATTAACGTATTCTAACAAACTCAACTAGCTAAGTGAAAAATTTTGATACAGTTGCCAATTTTACTCTGCATTGCATAAACAAATACTTCTCTATCTGATACAGATTTGTGGTGGTGACAAACCATTTCTGGCCCCAAATGACTTGCAGACCAAACACCTGCAACTTAAGGAAGAATCTGTGAAGCTATTCCGAGGGGTGAAGAAGAcGGGTGGGGAAGAATTTAGCCGGCGTTACCTGCAGCAGTTGGAGAGTGAAATAGATGAACTTTACATCCAATATATCAAGCACAATGATAGCAAAAATATCTTCCATGCAGCTCGTACCCCAGCCACACTGTTTGTAGTCATCTTTATCACATATGTGATTGCTGGTGTGACTGGATTCATTGGTTTGGACATCATAGCTAGCCTATGCAATATGATAATGGGACTGACCCTTATCACCCTGTGCACTTGGGCATATATCCGGTACTCTGGAGAATACCGAGAGCTGGGAGCTGTAATAGACCAGGTGGCTGCAGCTCTGTGGGACCAGGTAAGAACACCTTTAATTCACAACTAAATTCAGCACACATTTGAATGTCATCCATGTGCCAGCCACTGCATTAGATGTTGGAAATACAGATCAACAGGAATTGGGCCCCAGTCATCAAGAATGTTATGACCTGCCCAGATACAAGCAACTAACTATATAATACTGTGTAAAATGTGAAGTCCTACACAACTATTTTCCAGTTAACATTTCAGCATTTAAAAAAATATACATCAGTTTAGTAGTATATATACGCTTGGTGAGGGGCATATGTGAACCACAAT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59844 | GGT GGT GAT AAA CCA TTT TTG G | Wild type Forward | A | |||
| 59845 | GTA ACG CCG GCT GAA CTC TT | Wild type Reverse | A | |||
| 59846 | CAG ACC AAA CAC CTG CAA CT | Mutant Forward | A | |||
| 59847 | TCT ATT TCA CTC TCC AAC TGC TG | Mutant Reverse | A | |||
| 59848 | Fluorophore-1 | AGC AAG CAC CTG CAG CTA A | Quencher-1 | WT Probe | ||
| 59850 | Fluorophore-2 | AAG AAG ACG GGT GGG GAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59844 | 0.40 uM |
| 59845 | 0.40 uM |
| 59846 | 0.40 uM |
| 59847 | 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.