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Notes
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= 128 bp
Wild Type = 124 bp
Sequence
Wt Sequence (deletion in UPPER case):
tactgtttggaacctacttcctgactgcttccttcatcagttttttttcactttcattacttaattcctcttcatcctttagttagctgctaagttaccatactcttggaaacttaatgcttttagagtagtttcattgtgggtatattgatttgattacttattttaatacttgtcacctggtcattcaactggaaatcaactgaaaatgttcataacccgtttGTTGCCAATAATTATGTATTAAACACCTAGCACAAAGCAGACATTCCACAAATAAATACTAAGTGTGTTTATAAACCCACTTACATGATCTTATATATTATGATTTCACTGAAATAACTTGGTATAACATGAATTTTACTGAACGTATCTTCCTTTTCTCTGTTTTGTAAAAGTTATATTTTTACTAGATCTAATGTCAATAGTTCGGATCTAATTTAATTCTCTGTGTCTCCTTCCTCCCACAATATGCTGTAGTGGTTCCAGTCATAATGGCAAAAGACAATGATGGTAAAATGCCTGACGAAGATGCCCTGGAGGAGGATTCAGATAGTGCTAGTGAGCTAGGAAGTGATGAGGAATCTGAAGATGAAATCATAAGTGATGGTAAAACCTCAGCTGATGAGGATGAAAGTGAAGAAGAAGACGAAGAAGAAGAGGAAGATAGTGAGGAGGAAGAAGAGGAGGAGGAGGAGGATGAAAGTGACAGTGGTCCTGATCTTGCAAGGGGTAAAGGAAATGTAGAAACTAGTTCTGAAGATGAAGATGATTTGGCAGATTTGTTTCCCGAAGAACCTGGTTTTGAACATGCTTGGAGAGAGTTAGATAAAGACGCTCCTCGGGCTGATGAGGTAACACTTGCAATAAGATTGTTTACGAATAGCATTACAATTAGCTTTtatgggtgctggggttgatgtcattaaattaagcagaaaggaactggagggctagttcagtgctgcaattcttagctagtgtgggtgggctaacgtggggcatggtggcacacacctagagtagcatcactgaggaagcaggaaggttgctgctgtaagttttagggtcgccctgacttattgagttctaaactgacttggagtataaacctgctcagtgaagccctgtctcagaaaagaaaactaaacagaaagaaaacactaaaaatatattaagtgagaaaagctttatcggtgactgtttgttgttgattgaacccaggacctcctatctgtcagggaagatctctaccagtgagccacatcaccagaccctgtggtatccatgattaagattacaatgtgaggactggagata
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GCATTACAATTAGCTTTTAT and ACATAATTATTGGCAACAAA. This resulted in a 677 bp deletion of region Chr2:140,164,161-140,164,837 (GRCm38/mm10) and removes exon ENSMUSE00000557773.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 75993 | TAC TTG TCA CCT GGT CAT TC | Common | A | |||
| 75994 | ACA CAC TTA GTA TTT ATT TGT GG | Wild type Reverse | A | |||
| 75995 | AGA ATT GCA GCA CTG AAC TA | Mutant Reverse | A | |||
| 75996 | Fluorophore-1 | CGT TTG TTG CCA ATA ATT ATG T | Quencher-1 | WT Probe | ||
| 75997 | Fluorophore-2 | CGT TTT ATG GGT GCT GGG GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 75993 | 0.40 uM |
| 75994 | 0.40 uM |
| 75995 | 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.