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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= 103 bp
Wild Type = 120 bp
Sequence
Wt Sequence (deletions in lower case):
TGTGGTCTAGGGGCATGTATATTCCCACTCCCCTTTGTTCTTATGGCCAGGAGATTAAGAGCACAAACCTTTGGCTTAAGTTAAGGTGATGTACTTCACCTGCTTCTATCCTTAAGGAAAACTCGGAAGTTAGAGATTTTGTCCTAGTTGTCTGGTGCATGCCCTGAGGAGAGATCAAGTCTGACTGTGCCTCAGTTTTGTCCATGAATTGAGCATGTATTTTCAATTGCATAGTGGAAGAGTCTCACCTTGTTTTGGACACTCTCTTAGAAAGAACTGATCTGTGTTCCGGGGcttatttggtgcactcctgtctagaggagtcatgagccttgtgtcccaccaatgttgttgctgtcacttgctattctaattggaaagttgacattggctcaaaggctggcttaccacagtagaaattccattatgtaatattggttttgggtgctttttcagaggtcaattagaaactccaaactatttagaagaccctgccagccagaaagccattagtcctatcccaaatgaagtaatttgtccagaaagcccaggtaatttgtggaggaactatgaatttctggtaaatcgaatgtgctacttggagaacctcattcagtccttgaagatgaacatctttcacctgcaaactgaaaaggaatcgaatccacagaaaacaggtgtgcatggagaggcttgcggaggctctgcttctcggtgggtgcttggtgggcccgtgaatctgtgcttcattcacctttccactgctgtcttctgtgctgtgcatagcttttctgaacgatcagctgaatataatacaaggggagcattccaagggcctgaagctgttgcaacttgaagtgatgaatttgcgccagcaattgaaagttgtcaaggaggaagaagacagggcacaggatgaaatgcagagattgactgccactctggaaattgccacggagacaaaggttcgtggtcagtgttctgtagtgcattgtccctgcTCTATCTCGACTAGAAGAGACAACAGCCCCATAAAACTGAGTCACAGATGGGTGGTGTTGATATTAGGTTCTTGACATTTGAAGCTTGTCTGCCCCGATAACCTCCACTAGTGTATGTTGTCAGTGGTAGTTCACAGACTCCTTGCTGTGTATAAAAGATAGTGAGCATCTAGACTCTGCCCTCTTTAAGAGCTACACTGTAGTTGGGAGGTCAAGGCATAACGTAAGTTAAAGAGGAAAAAGCCAAAGTAGAAAGCAGAGAACAGTAAGTGCACAGGAAGTGTGTTCAGGCACACTTGAGAAGAGACGAACCCTCAGCCAATGTAGATCTGAAGGTGCAGTCCCTAAGGGCATTTCAGGGAGTACACTCACTGGCAGGCAGCAAAGAAGCCATATTGCTTAGAAAGGAAGCCAGCAGCATTGTTCTGTTGTCATGGACCAGGCAAGCTAATAGATTATCAGGTTATTTTGAAAGTCCTCAAGAAGTAGCATAATATTAAAGCAGTCTATTTTAGTAAGAGTAATGGAGCTGAAAAGTGTCTTGTTCAAGATCATCACAGGCTTGCCCTCTTCCTGTAACT
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GGAGTGCACCAAATAAGCCC and CTTCTAGTCGAGATAGAGCA. This resulted in a 707 bp deletion of chr1:54,263,282-54,263,988 (GRCm38/mm10) that removes exons ENSMUSE00001296518 and ENSMUSE00001220531.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 75766 | TGG ACA CTC TCT TAG AAA GAA C | Common | A | |||
| 75767 | AAT TAG AAT AGC AAG TGA CAG C | Wild type Reverse | A | |||
| 75768 | ATA TCA ACA CCA CCC ATC TG | Mutant Reverse | A | |||
| 75769 | Fluorophore-1 | CTA GAG GAG TCA TGA GCC TT | Quencher-1 | WT Probe | ||
| 75770 | Fluorophore-2 | CGA CTA GAA GAG ACA ACA GCC C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 75766 | 0.40 uM |
| 75767 | 0.40 uM |
| 75768 | 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.