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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= 78 bp
Wild Type = 94 bp
Sequence
Large deletion. Mutant sequence with junction in uppercase:
ggttaggagcaccggctgctctgtgagcagatctgggttctgtaactccagccaggttgaggtatccgattcccttctttgactctgtgggcccaacaaccacgagatgtgcagacacacaggcagtgcactcatacacagaagcaaaaataaaacaaatatcgAGaatggaggccttttttggcttgcttatagggttcaccacatgtggagttgcttgggcctgtctcactcatttggtctgctcactgtgttcccccgtgtgtcagatggagggctagcctcggctcactgtgttcccccgtgtgtcagatggggagctagcctctgctcactgggttcccctatgtgtctgatggggggctagcctctgctcactgggttcctccgtgtgtcagttagagggatagcctctgctcactgggttcccctgtatgtcagatttggggctagcttctgctcactgggttcccctgtgtgtcag
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: CCCCAACCAAAAATTTAGGG and TGGTTAGCCCGGATTTGTGC. This resulted in a 1,277 bp deletion of chr17:27,879,102-27,880,378 (GRCm38/mm10) that removes exons ENSMUSE00001293900 through ENSMUSE00000449238.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 76054 | GCA CTC ATA CAC AGA AGC AA | Common | A | |||
| 76055 | TGC TTA TTT TCC TTA CTT CTC TG | Wild type Reverse | A | |||
| 76056 | GTG GTG AAC CCT ATA AGC AA | Mutant Reverse | A | |||
| 76057 | Fluorophore-1 | ACA AAT ATC GAG AAA ATC CCC A | Quencher-1 | WT Probe | ||
| 76058 | Fluorophore-2 | ACA AAT ATC GAG AAT GGA GGC CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 76054 | 0.40 uM |
| 76055 | 0.40 uM |
| 76056 | 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.