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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= 83 bp
Wild Type = 106 bp
Sequence
Wt Sequence (deletions in lower case):
CTTGCTTTGGAGATAAGAAAATCTTAATTTGTCACTTACCTGTGATACTCTGTAGAGCTTATGATGGTTCTCGTTGGTTCTCATTGTTATGTGGTGAATGGGAGCACTGACCTTGGCTTCTGGACGTGTCCTGAGATGACAGGATCCTGGGTGTTAAAAGGAAGGGAGCCGTTCATTCTCTGGTTCAGTGAAGCCTTAGTCTGTGGTTTAACTGTATAATCCAACATCACCCAACAGAGCATTCACACGCTCTACCTCTCTCTCTGAATTCTCTTTGTGTTTCTAGGAGACAAGAATCCTGAGGAGATGGAGTCTCTTGAGGAAGTGGGATTGAGGCACCTGTCACATGAGGCGCTTTTCTGCTCACAAATCTGGCAGCAGGTTAGCAGGGACctaatgaaggctggagactgcagagtaagcatcagggaaactggctctccgttgaagcgagatgatgcacatggtgcagacaggaagtacagcaaacattccggtcagaaaccatggctccaactccactgtggaacagatggtggtgaagaaccttacagagaggagaggagcgagaaagactcttgggactctcctctgcaggctaacgggcgaccccccgcaggagagaagaggtataggtgtgaaaaatgtgaccacgccttctgtcggttatcgggtctccaagcccatcaggtgaggcacactggagagaagccatacaagtgtgaggagtgtggcaagggcttcactcgggcctccacccttctggaccatcagcggggccacactggaaacaagccctatcagtgccatgcttgctggaagagtttctgtcacagctcggagtttaacaatcacataagagtccacacgggagagaaaccctatgtatgcgaggagtgtgggaaaggcttcagccaggcctcccatctcctggcccatcagagaggccacactggagagaaaccctacaaatgtagcacgtgtgggaagggcttcagccggagttcagatctcaacgttcactgcagaatccacacgggagagaaaccctataagtgtgagacgtgtgggaaagcctttagtcgggtctccatactccaggtgcaccagagggtccacagtgaagacaaaccgtaccagtgcgcagagtgtgggaggggcttcactgtagagtcacaccttcaagcgcaccagcgctcccacacgggagagagaccctatcaatgtgaggagtgcgggcgaggcttctgtcgggcCTCCAACTTTCTGGCTCATCGTGGAGTCCACACAGGCGAGAAACCGTATCGATGTGACATATGCGGAAAACGCTTCCGGCAGAGGTCCTACCTCCATGACCACCACAGGATCCACACGGGAGAGAAACCTTACAAATGTGAGGAGTGTGGGAAAGTCTTCAGTTGGAGCTCATACCTCAAGGCCCACCAGAGAGTTCACACAGGAGAGAAACCATACAGATGTGAGGAGTGTGGGAAGGGTTTCAGCTGGAGCTCAAGTCTCCTAATTCACCAGCGCGCCCATGCAGAGGATGAGGGTCGCAAGGATTTGCCTGCATCAGAAGGCTCACAGGGGAAACAAACTCTATAATGTTTTATAGTTTGATATTTCAACTGGAAGCTGAAAGGTCCCAACTCTTAGAGTTCTCAAGGGAGTTG
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GTGCGGGCGAGGCTTCTGTC and GGTTAGCAGGGACCTAATGA. This resulted in a 863 bp deletion of chr7:24,302,904-24,303,766(GRCm38/mm10) that removes exon ENSMUSE00000498089.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 75722 | CTG CTC ACA AAT CTG GCA | Common | A | |||
| 75723 | ATG TGC ATC ATC TCG CTT C | Wild type Reverse | A | |||
| 75724 | ATA CGG TTT CTC GCC TGT | Mutant Reverse | A | |||
| 75725 | Fluorophore-1 | CTA ATG AAG GCT GGA GAC TG | Quencher-1 | WT Probe | ||
| 75726 | Fluorophore-2 | TTT CTG GCT CAT CGT GGA GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 75722 | 0.40 uM |
| 75723 | 0.40 uM |
| 75724 | 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.