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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= 120 bp
Wild Type = 145 bp
Sequence
Wt Sequence (deletion in UPPER case):
atctctgaaccccaggaggggagactgaagagggcaggcttgttagacaatcacagctgggggcaggggtaaatttagcctttgttcagcccccagttatgtgagggatgcaggactcggggtagcaggagggaagggaacaggtggcatgggattaaccctgttcccaagaaaccctccccctgtaattcctcttgggattctcccaaggcccagagctggggacaggactgaagtggcccggctccccgccttcatccctttgggtccctccccagaagtggctgccagttGGTTGTGATAGAGAGGGACTGAATAAGCCCTGTCATCTCCTCACCTGCCCTTTTTGTCCCCTACAGATCGAGTTTAACAAGGACCAGCTGGAGGGTGAGGAGAAGCCCATTTCCTAAGCCCCATTGTCAAACCCCAAACTCCTGGTCAGATTCTCTGTTCTAACGACAGTCCTGACCTTGAAACCTCCCACACTTGTCCTGTTCTCCTCCCTCCCATGCCTGGCTCTAGCACATGAATTTCTGAAGCAAACCTCCCTCTCTCTGTTGGTGATGGGGATCCTTGGTCACACTGCCTTCTCCTTACATCTCAGAATTCAGAGAGGCCTTTGAGCTGTTTGACCGAGTAGGTGATGGCAAGATCCTGTACAGCCAGTGTGGGGACCTGATGAGGGCCCTGGGCCAGAACCCTACCAACGCCGAGGTGCTCAAGGTCCTGGGGAACCCCAAAAACGAGGGTGAGAGGAGCCTTGAGTTTTGTGGGGGTTTCTGTTTTCTTCTTGGTGTTGAACGTTGAAtacagggttctgcctgggccattgagctacatttctagccagcaactttctcttcttcctctttttcttctctctctctctctctttctttcaagataggatttcagtatcccagccaagaatgaccttacattctgattctcctatctccacctcttgctactgcccactagagtggtgggattactccagtttatgtagtgctgggttgaatccagggcctcaggcaagccaggcaagcccttattacagaggtactgtttttttttactttttgagacaggtgctcaccaaattactcaggttggccagggacctacagttctatcccagcagccccaatcccc
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GGTGTTGAACGTTGAATACA and CCCTCTCTATCACAACCAAC. This resulted in a 515 bp. deletion of Chr10:128,496,193-128,496,707 (GRCm38/mm10) and removes exons ENSMUSE00000150202 and ENSMUSE00001067623.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74718 | GAG GTG CTC AAG GTC CTG | Wild type Forward | A | |||
| 74719 | AAG TTG CTG GCT AGA AAT GT | Common | A | |||
| 74720 | GGG ACA GGA CTG AAG TGG | Mutant Forward | A | |||
| 74721 | Fluorophore-1 | TCT TGG TGT TGA ACG TTG AA | Quencher-1 | WT Probe | ||
| 74722 | Fluorophore-2 | AAC CCT GTA AAC TGG CAG CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74718 | 0.40 uM |
| 74719 | 0.40 uM |
| 74720 | 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.