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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
>chr3:8515051+8515188 138bp TGGATTACAGAATATTTCACTTCCAA AGGCCTTCTGTTGAGTCCTG
Mut= 89 bp
Wt= 138 bp
Fam=Mut
Hex=Wt
The WT Probe (primer 64602) anneals over the nucleotide sequence containing mouse genomic variations rs37789913.
Sequence
Wt Sequence:
atgagcatgtgcccctcagaggaagtccactgtcctgatgagcatgtgcccctcagaggaagttccactgatgatgagcatgtgcccctcagaggaagttccactgtcctgacttttaaatctgtggcaatggattacagaatatttcacttccaaactcatatgcatagggatttgattcctctgaggatatgtattctgtgccatcactctctcttaattagattgtttaaaatatatttatattacaggactcaacagaaggcctttgaggaaaatggtgggagaaataagcctggctctgtgaatatgtgtgtgggtgggtgggtctgtgactcagttaagaaaaataggggactttcaggatagcatttgaaatgtaaatgaagaaaatatccagaaaaaattgttaaataaaaaagaagggataatatataatatgatattactgtcacttacaaatgatacacagaacagaagttaaaaccagaacctt
Mutant Sequence:
GGAAGTCCACTGTCCTGATGAGCATGTGCCCCTCAGAGGAAGTTCCACTGATGATGAGCATGTGCCCCTCAGAGGAAGTTCCACTGTCCTGACTTTTAAATCTGTGGCAATGGATTACAGAATATTTCACTTCCAAACTCATATGCATAGGGATTTATTACTCTGGGAATTATGTGTTCTGCCCCATCACTCTCTCTTAATTGGATTTTTAAAATTATATTCATATTGCAGGACTCGGCAGAAGACCTTCGAGAGAAAGGTAGAAAATAAGAATTTGGCTCTCTGTGTGAGCATGTGTGCGTGTGTGCGAGAGAGAGAGACAGACAGCCTGCCTAAGAAGAAATGAATGTGAATGCGGCTTGTGGCACAGTTGACAAGGATGATAAATCAATAATGCAAGCTTACTATCATTTATGAATAGCAATACTGAAGAAATTAAAACAAAAGATTGCTGTCTCAATATATCTTATATTTATTATTTACCAAATTATTCTAAGAGTATTTCTTCCTGAATACCATGTGAGAAAATTCTTAAGAATTTATTGAGTATATTAAGCATTCACTGGGTTATAAGGAATTCACTAGGTATGACCCTATTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50411 | TGG ATT ACA GAA TAT TTC ACT TCC AA | Wild type Forward | A | |||
| 64599 | AGG CCT TCT GTT GAG TCC TG | Wild type Reverse | A | |||
| 64600 | TTG GCT CTC TGT GTG AGC AT | Mutant Forward | A | |||
| 64601 | ACA AGC CGC ATT CAC ATT C | Mutant Reverse | A | |||
| 64602 | Fluorophore-1 | CTG TGC CAT CAC TCT CTC TTA ATT AGA | Quencher-1 | WT Probe | ||
| 64603 | Fluorophore-2 | AGA CAG ACA GCC TGC CTA AGA AGA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50411 | 0.40 uM |
| 64599 | 0.40 uM |
| 64600 | 0.40 uM |
| 64601 | 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.