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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
Sequence
Large deletion: Mutant sequence with junction in uppercase
tggggccctcctgctgagagccgggcttcctggttagtggaactagcgaggcggggccagtatttggccggaaagtaccttaaggaaccccaagagggtaagatcggggactcacgaagtcttcccatttttctattgcagggaggggaactacatgcccagcataggtcctgaaccatGGtcaacctcttacagcacaagcctgcacatgctggacactagccagtgggcccaggcaatcggaactgttgaagaaaacaggaaggatgtcaggtgacttgtccccaagctggttcaccccaggagctagggaggctgatgcagtggcttagctctgagctccac
This mutation is a 1236 bp deletion of Chr2:25,194,651-25,195,886(GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 70073 | CTG GAC ATG AAT TTG CCA TC | Wild type Forward | A | |||
| 70074 | CTA GTG TCC AGC ATG TGC | Common | A | |||
| 70075 | TAT TGC AGG GAG GGG AAC TA | Mutant Forward | A | |||
| 70076 | Fluorophore-1 | AGC AGG TAG TAG CTA AGG TC | Quencher-1 | WT Probe | ||
| 70077 | Fluorophore-2 | CCT GAA CCA TGG TCA ACC TCT TAC AGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 70073 | 0.40 uM |
| 70074 | 0.40 uM |
| 70075 | 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.