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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= 73 bp
Wild Type = 96 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase
cagccatggcacaggagaaacagttctacctcttcacccaaaggaagccgggagcagactgtttcctccgtggctaggaggaaagtcataaaacctacccatatagtgtcacaccacctaccccacaaggccacacctcctaatagtgccactccctgggccaagaatattcaaagcagcacactggccctgtgtgaggcaaggcttggttgctaagtgaccatcccagggcagccacccactgtccacttgctatgcccactcagcaatagctaccctgatactgctcattcctttgccaCCctctgagcttgggcctggcagtcacttaggaaccagaagaagaaataagaggatttctggtggagcctcattgctctgttcgtgcgtgtgcgttcgagaatgcgttcgagactctctctctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttctccct
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GTTGAGCTGCCTCGACATGG and CTCAGAGGGCCAGATCTCGA. This resulted in a 1,875 bp deletion of Chr6:29,471,525-29,473,399 (GRCm38/mm10) that removes exons ENSMUSE00000898298 and ENSMUSE00000890596.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76562 | ATA GCT ACC CTG ATA CTG CT | Common | A | |||
| 76563 | TGA GAT ATT CCT CCT TCC AGA | Wild type Reverse | A | |||
| 76566 | TTC TGG TTC CTA AGT GAC TG | Mutant Reverse | A | |||
| 76567 | Fluorophore-1 | CAG CTC AAC ATG GAC ACC | Quencher-1 | WT Probe | ||
| 76568 | Fluorophore-2 | TTT GCC ACC CTC TGA GCT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 76562 | 0.40 uM |
| 76563 | 0.40 uM |
| 76566 | 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.