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Notes
Taqman Probe/Endpoint protocols use a different analysis than qPCR. Please see your equipment manual for more information about Taqman endpoint analysis setup.
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
Mutant= 99 bp
Wild Type = 114 bp
>chr13:55414699-55414812 114bp GGCATCCTCAATAAGACAGT AGTTCACGGTTTATTCTGGT
Sequence
Large deletion: Mutant sequence with junction in uppercase
catttcctcctacatgctacttttagaaccgcccccgaacccccaccaactctaccgccctctccttctcctcctagttagatgttctgtgacgtttgccttacgtcacagccccgccctgcgaacttcgggcatgctgccagtgttcttagctctgaatgcagccggccaccccccctaatgcaagcacaagttgctgggactgactgagcggagatgagtgactggaagggctacatcaGCtagaccgcccgtggttggtagggagaccagaataaaccgtgaactgggtgctctgagaggtatcctctgtgtatttgtgaggagttgcaaacaagaaaactagcttatggggagattcttgcagaacaaaataatcctggtggtcct
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GCGTGAGCAGTGCTCTTAGT and CTACATCAGTGCAGTGCTGC. This resulted in a 396 bp. deletion of Chr13:55,414,745-55,415,140 (GRCm38/mm10) and removes exon ENSMUSE00000352515.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 75299 | AGT TCA CGG TTT ATT CTG GT | Common | A | |||
| 76276 | GGC ATC CTC AAT AAG ACA GT | Wild type Forward | A | |||
| 76277 | Fluorophore-1 | CAC GAT CTG ATT GGT GGG | Quencher-1 | WT Probe | ||
| 76764 | CAA GTT GCT GGG ACT GAC | Mutant Forward | A | |||
| 76765 | Fluorophore-2 | AGC GGA GAT GAG TGA CTG GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 75299 | 0.40 uM |
| 76276 | 0.40 uM |
| 76764 | 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.