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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= 104 bp
Wild Type = 106 bp
Sequence
Large deletion: Mutant sequence with junction in lowercase:
CACTTCCTGCCAAATGCATTCCTTCCCACATAACTTGAGAGGATTGGATCAGTCCAGGAGTCAACATCAGCTCTGACTCTCAACTGGTCAGGACTTACAGGCGTCCTCACTGTGAGTAgtCTGGGAGACAGTACAGCAAGGCACCCGTATTAGTCACGTATCATACCATGACCAAATGCCCAACAGTAACAAGTCTACAGAAGAAAGACTGCCTTTGGCATATGGCTTAGAAGGCTCAGACTAGAATTCCTTAGCCCCAGGGGCTTGTGCAGAACAGTATGTCAAGCGGAGAGAGTGGA
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: CCATGTACAGCTGGCAAGTC and CGTCCTCACTGTGAGTAGCC. This resulted in a 1,688 bp deletion of Chr7:24,104,406-24,106,093 (GRCm38/mm10) that removes exon ENSMUSE00000990787
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 79839 | ATG GGA TGG TTC TTC ATT GA | Wild type Forward | A | |||
| 79840 | GAC TAA TAC GGG TGC CTT G | Common | A | |||
| 79841 | GTC CAG GAG TCA ACA TCA G | Mutant Forward | A | |||
| 79842 | Fluorophore-1 | AGA TTG TCT GCT TTG CGG | Quencher-1 | WT Probe | ||
| 79843 | Fluorophore-2 | TGG TCA GGA CTT ACA GGC GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 79839 | 0.40 uM |
| 79840 | 0.40 uM |
| 79841 | 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.