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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= 69 bp
Wild Type = 117 bp
Sequence
Large deletion: Mutant sequence with junction in lowercase
GCCATGCAAGGCCTAGCTTCCAAGCCACCATGCCAGCCAGCAGGAGGGCAGAGGGCTGGAAAAGGACACCCTCCCCATCCTGTTCCCTCCCTCAGTGGAGAGAGTCAGGGGTCAAAGTCCAAAGTTCATTCACTGTCAGGCCTGGTACAGGGGCCAGCAAGCACAGCAGATCCCCCACAGCAGGGTACCTTGTTAAAGACCATGGCCACCagATCCACCTCTGCTGAGTCTCCTCCCCTACTACCCTTACTGTCATATTGACAACTATGGGTCCCAGTAAACATCAGAGTTCAGCCCACTGTTCTCTTTGGCCTCTTTGGTTCCTTGTCCCCACCTCTATGTCGTCACGCTTCAGCTACACACTGAGTATGACGCCATAGCAGCAGCGTGCGAGCTCATACACAAACCAGCCCCCTGAAAGCTATCGCGAGATCACTCGTTACCACAACTCCCAGAAACCTCTAGGCAGCAGTGAGCCCTGGGAGAGAACGAGAACTGGCTGCTTTGTCCGATGGGAGTT
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: AACTTGAGGGAGCGTACTGG and ACTCAGCAGAGGTGGATCCC. This resulted in a 2,189 bp. deletion of Chr7:44,465,482-44,467,670 (GRCm38/mm10) and removes exons ENSMUSE00000795968 through ENSMUSE00000277140.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 75210 | CAG TGC TAT CCC TCC AAA C | Wild type Forward | A | |||
| 75211 | GGC CAC TTC TAC CAC AAG | Wild type Reverse | A | |||
| 75213 | TGA CAG TAA GGG TAG TAG GG | Mutant Reverse | A | |||
| 75214 | Fluorophore-1 | AGA CCT GAC TCT TCT CCA AG | Quencher-1 | WT Probe | ||
| 76515 | Fluorophore-2 | CAG ATC CAC CTC TGC TGA GTC | Quencher-2 | MUT Probe | ||
| 76516 | CTT GTT AAA GAC CAT GGC CA | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 75210 | 0.40 uM |
| 75211 | 0.40 uM |
| 75213 | 0.40 uM |
| 76516 | 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.