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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
>chr13:56176178-56176268 91bp TCCCTTGAGACCTTTGTCTGC TCCCGGTTCATTCATCTGTC
Mut= 99 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case;and insertions with carrots ^g^ (ggctg insertion):
CTTTATTCCTTGCTGAGAAGGAAGGGCACACCTGTGTTATAAAACAATACAATTTGGAGAGGATGGGCAGTTATGTCCTGTTGGAGTATAGGGAGATGGAATTCCAAGTTTAGTGTCAACCTCCACCCCATCCCAGCTTGGGGAGAATAACTGTATCTATTTACACATGGTCCTCATCAGCAAGATGTGGCCCCAAGATCTCTAAGTAGGCAGCTTGCCTGGGCTCAGAAAACTACAGAAGTGTGTCTTTGTCATGTTCTTCCCAGGATCTATGGAGAGGCCCCTCACAGTCCTGCAGGTGAGCCTGTACCACCCTA^cacagggcccggtcgcctttgcccacgtcccacagcagctgcaacatgacgccagccggctgttggttgggcgaggacagaacacccatctccagctgcagctgccacagttgtctcgctaccatttgtccctggaaccctacctggagaaaggcagcagtctgctggccttctgcctcaaggtgctgacccgcaagagctgtgtgtgggtcaacgggctgccactgaggtacctggagcaggttcccttgggcaccatcaacagaatctccttctctggcatccagatgctagtccgcaaagaggggggagcctcccttgagacctttgtctgctacttccatctcagcccttcacccctgatttac^AGACCCAAGGCTCAGGAGACAGATGAATGAACCGGGAAAACTCTGTTAAGAAAGAGACTCCCCCTGTTTTGGGGGAGGCAACTCATGAACTCTCGAGGTCTCTCCACAGCCCCTCCCAGTCTCAGACAGCTGCTTCCCTGCCAAGCCCGGAAGAAGCAATAGAAACAAAGTTGCAGAAGGGGCCCTCAGACAATGCACCCCACTAGCCCTGCTGCTTGAGAGCAAACCTGACAAAAATAGGGTTTGAGATGGTAGAGCTATATCCTGTGAGGTTGACACACAGAGTTCACATGTAAAGCCTGCTGGCCGCCATGTTCAC
This mutation is a 368 bp deletion beginning at Chromosome 13 position 56,176,215 bp and ending after 56,176,582 bp (GRCm38/mm10). In addition, there is a 5 bp insertion (GGCTG) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52824 | TCC CTT GAG ACC TTT GTC TGC | Wild type Forward | A | |||
| 52825 | TCC CGG TTC ATT CAT CTG TC | Common | A | |||
| 52826 | TCC CAG GAT CTA TGG AGA GG | Mutant Forward | A | |||
| 52827 | Fluorophore-1 | TCA GCC CTT CAC CCC TGA | Quencher-1 | WT Probe | ||
| 52828 | Fluorophore-2 | ACC ACC CTA GGC TGA GAC CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52824 | 0.40 uM |
| 52825 | 0.40 uM |
| 52826 | 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.