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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
>chr9:71548658-71548739 82bp ATTCTGACAAGAACAGGTGAGG CACACACACAACCAGTGTGC
Mut= 82 bp
Wt= 82 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (A insertion):
GGTTTGCCTAGAGTGTTTTTTTCCAGATCCATACATATGTCTATGCATTGGGCCCCTTTGAGTTCTGAGCTCTGACCTAGTGGATGTGTGAACAGGGAGTGTTGTACAGACTTGGAGAGCTGGGTGTGCCTGGTG^ttcatcctctgcctagctccatctggatccattgattatgtctgttttaggaacggcatcagctccagctccagctccttgagcatgagacagaaatgtcaggggagatggcagattctgacaagaacaggtgaggcaggctggccttggacttcgagcaactcag^AGGTGGATATGCACACTGGTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATGCGCGTGTGCACTAGATGCACATGTGTGCATGTTCTTGCCCACAGAGGCCAGAGGAAGAAGTCTGGTGTCCTTCTCTTATCACTTTCTACCTCATTCTCTTGA
This mutation is a 166 bp deletion beginning at Chromosome 9 position 71,548,688 bp and ending after 71,548,853 bp (GRCm38/mm10). In addition, there is a single bp A insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42924 | ATT CTG ACA AGA ACA GGT GAG G | Wild type Forward | A | |||
| 42925 | CAC ACA CAC AAC CAG TGT GC | Common | A | |||
| 42926 | TGT GTG AAC AGG GAG TGT TG | Mutant Forward | A | |||
| 42927 | Fluorophore-1 | CTG GCC TTG GAC TTC GAG | Quencher-1 | WT Probe | ||
| 42928 | Fluorophore-2 | TGG AGA GCT GGG TGT GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42924 | 0.40 uM |
| 42925 | 0.40 uM |
| 42926 | 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.