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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= 109 bp
Wild Type = 129 bp
Sequence
Wt Sequence (deletion in UPPER case):
gggatagcattgaaaatgtaaatgaggaaaatacctaaaaaaaaaaaaaaaaaaaagatagaaacatctccttggatatgcttagtatcttcagtggagtccctatatgagtaacataggtccacttagtctacagagtacagcatagtgtggaaacaatcctagagaagcagcctgcaggggaaagaatgtcaagctgtaggatttggaaactaaccttgggcaggctaacgcaggttttataaagaaaccattttgaagcttctacATCTTGTGGATGGAGCATATGTGGAAAATATATCTGACTTAAGTCAGTGTTTAACATGAAATGCAGCAGGGAAGGAAGGCAGCCATGTTCCCAAGGGGTTAAGGCCCACCAAGGCTAAGGCTTCCCCTGACGGAAGGGACACAGCGACAATGCCCATGCACAGGAACACTCAGCGCGTGTGAAAACGAGTGACAGGCCAAGGAACAACAGTCTAGAGACTTGGCCTCATACCTCCATTTCGGTTCTGGCTCTTGATTTTTCGATAGTCATTGTACAAAGGCTCCAAGTACTTGTAGCAATCAATTGCAGTTCCTGTGAGCCGCATGTAAAGTGCCCCCAGCATCCGGACATACCTACCAGAGAGAGTACAGTGAAACAGCAGGCCATTTCAAGACTTCCTGAGTTTTATAACTGGCTTTTCTCATGACAACAGTCGGTAAAAATGTTTCTTGGTTTTTTTTTTTCTTCTTGTTATCAGATAAAGCTGTAATTCTGAAGATATCGTAGAACAGATGATGGCTAAAATATGGAGACCTAGTCAACTTCCTAATCTACCGATCACGAGCTGtgagggcttgctacatgttgttcaactttcaggtatatcatctgtaaaactttacagaattagcctaagattccaaaaaggtaacacttacaacacagtaaacataatatatattttataataatgctagaacatgcatacataggcacaaatgtaggttctagataaaggtcaaagtcatagaggttaaaattctgatttcataaattactaactttagtaccagatattgaacgtgttttctcaacctctaaaatggagaat
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: AAGCTTCTACATCTTGTGGA and CTACCGATCACGAGCTGTGA. This resulted in a 568 bp. deletion of Chr4:108,575,138-108,575,705 (GRCm38/mm10) and removes exon ENSMUSE00000180481.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74711 | TTT GGA AAC TAA CCT TGG G | Common | A | |||
| 74712 | TGC ATT TCA TGT TAA ACA CTG A | Wild type Reverse | A | |||
| 74713 | ACA GAT GAT ATA CCT GAA AGT TGA | Mutant Reverse | A | |||
| 74714 | Fluorophore-1 | TTG TGG ATG GAG CAT ATG TG | Quencher-1 | WT Probe | ||
| 74715 | Fluorophore-2 | AGC AAG CCC TCA GTA GAA GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74711 | 0.40 uM |
| 74712 | 0.40 uM |
| 74713 | 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.