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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= 129 bp
Wild Type = 142 bp
>chr6:88892966-88893107 142bp ATTGAGAATCTGGAGGACCT GCTCCTTGAAGACGTTGT
Sequence
Wt Sequence (deletions in UPPER case):
acacagagaaatcctgtgtcaaaaaaagaaagagtattagtacactgctcttaagccccgaagcatctctagccccatacgtgttcacactgttgagagcagacttgggttagccagtcagtaccttgcatttcccctggggtcgtggaattctagagattttgcacgtttgttatccaaagagcgtgttctattgcttttattaggttttcatagtttctgttctgtgtcatgattcccccatttcctccctgacagtttcatgttttctagtgtctgatgaagatggtgccatttgaagattgtaaaaatgaccttgtttgatacttggttggtaattttctcaagattgggggagactgcatctagaggaagcactggccacaTCCATCATATACCACCAGCAGGTGGCGCTGTGGATGTGGGGCTTGCTCTCCACCCAGATTCACATGGCTGTGTACCTCCTGCACGGAAAAAGACTCCCTTTTCCATCCTGGGGGCTTCGGCCTCCTTGACGGAGTTCCTAAATCATATTCTTCAAACTTTCAACTTCCTGTTTGCAGCTGTCCAGCAAGGACTTTGTAAGCCCGTGGAGCAGACCTAATTCTTAAGTGGGTTGTTTGGGGATTCATTGTCCACTGTTGGTCTCTTGCAGAGACTACCGTCCCATTCCGGAGCTCGATGTCTACGAGGCCGAGGGATTGGCCCTGGATGATGAAGATGTGGAGGAGCTGACAGCCAGTCAGAGAGAGGCAGCTGAGCGGACCATGAGGCAGCGGGACCGTGAGGCTGGCAGAGGCCTGGGACGCATGCGCCGGGGGCTGCTCTATGGTATGTGTGGTGGGCATCCCAGAGGTGCCACTTGGACGTCCAAAGCATCAGGAATGGGGAAGACGTGGGCAGCGAGTGTTGTGTAGAGGATGCTGGGAGAGGCTTGCTCACGGGGGTGCTTTGCTACCCAAGACAGCAGCGAGGAAGATGAGGAGCGGCCTGCCCGTAAGCGCCGCCACGTAGAACGCGCCACAGAGGATGGCGAGGAGGATGAAgagatgATCGAGAGTATTGAGAATCTGGAGGACCTCAAGGGCCACTCGGTGCGCGAGTGGGTGAGCATGGCAGGGCCCAGGCTGGAGATCCACCACCGCTTCAAGAACTTCCTGCGCACCCACGTGGACAGCCATGGCCACAACGTCTTCAAGGAGCGCATCAGTGATATGTGCAAAGGTATGGTCCTGATAGGCTGGGGCGAGCAAGGCTCAGAGGTGCAGCATCTCTCTTGAGTGAGGTCCTGGATCAGTCTCAGCACTGGGGGGTGAAGGTGGCCTGGCTTCTGTATCCTGCTGGCTCTGTGTGCCGCACTGAACTCAGAATGCACAGGAGCATTGGGTGACAAGGACTGAACCTGAGGACCCCAAGGTCTCCTGTATCTGCACCAGAGCTAGGCTGTTTGGGGACATGTTTAAAAATGTATTACGACCCTGTTTtggtttttggagggtttggttggttggtttttgcagaatcaggttgagcctagggcctagcacattctaagccagtgttctcccactgggccactctccagcctgtgcatttcaactttgatgcaaaaatggcagtctttctgtgtgacagcattggctttacagggtatgacagcgtggggctgtggcaacccctccagcaatggctgtagttcttctgtagcttctctatggcctggggcacactgctctgtagcctgaggg
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: ACCCTCCAAAAACCAAAACA and CACCTGCTGGTGGTATATGA. This resulted in a 1098 bp deletion of region Chr6:88,892,685-88,893,782 (GRCm38/mm10) and removes exons ENSMUSE00000194955 and ENSMUSE00000194959
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74141 | ATT GAG AAT CTG GAG GAC CT | Wild type Forward | A | |||
| 74142 | GCT CCT TGA AGA CGT TGT | Wild type Reverse | A | |||
| 74143 | TGG TGC CAT TTG AAG ATT GT | Mutant Forward | A | |||
| 74144 | CCA ACC AAA CCC TCC AAA | Mutant Reverse | A | |||
| 74145 | Fluorophore-1 | CCG CTT CAA GAA CTT CCT G | Quencher-1 | WT Probe | ||
| 74146 | Fluorophore-2 | TGA CCT TGT TTG ATA CTT GGT TGG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74141 | 0.40 uM |
| 74142 | 0.40 uM |
| 74143 | 0.40 uM |
| 74144 | 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.