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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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= 102 bp
Wild Type = 78 bp
>chr1:165655620-165655697 78bp ACAGCTAGTCAGGGTCATC GCTTCCGCACCCATTC
Sequence
Sequence (deletion in UPPER case)
agtgttttgttttttttcccagaatacatagccctccacctggccttttaaattttttttatatttttttattaggtattttcctcatttacatttccaatgctatcccaaaagtcccccataccctcccccccactcccctacccatccactcccactttttggccctggcgttcccctgtactggggcatataaagtttgcaagtccaatgggcctctctttccgttgatggccaactaggccatcttttgatacatatgcagctagagtcaagagctccggggtacctttttaatttttaagataatgttccttcatatcttcttccagGTGCGGACTAGAGGCTCAATAAAAAGACGTCCTCCCTCGCGGCGATTCCGAAGGTCTCAGTCGGACTGTGGGGATTTTAGAGATTACAGGGCTGTGGAGCCATCTCAGGAAAACGGTGCCAGGGAAGAGAATGGGGATGACGTGTTTGCTAGCAAGAGCAAGGACCCCGGGTCCCCTCAACTCAACCAGGAGGCTATGGCAGACGGGGTGGAGGGAACTCCGTGGTCTGCAGAAAAGCCACGGAGAAGGAACACGTGTAACAGCACAGAGAAGCCAGAGGAGCTGGTCAGGACCCCAGAGGAGGCGAATGCTGGAGAGAAGGTTGGACAGAATCCAGACACAGCTAGTCAGGGTCATCCAGAGGTCCAGGCGCCATCGCAAACCGGCAGCCCAGAGGCTGAGAATGGGTGCGGAAGCCCACGGGAAGAGACAACCCCGGGAGAGCATACAGACACTGGGAAGGCCACTGAAGGGACAGCCTCTGAGGAGAGGGTAGCAGATGAAGATAGGCTCGGACAGAAAAGCCCAGACGCAAATATGCCTGAGGAGGAGGGAGTGGTCAGGGAGAAAGCCCCACAAACCTCTTCTGGAAAAGCAGAAGGTACTACTATCGCAGAGCCGGATACAAAGCAAAAGGAGGAGGCGCCTCTGGAGCCAAGCTGTAGCCCAGGGGCTGACCATGCTGCGGGAGAGATCACCAGTGAGATCCAGAATGAGAAAGCAGTCTCCATGgtaggtggccctgatggtttatgctgtatcaggaccagagctgcccctatcagctctgtatgcagaagcaaagtcaacagcgccctctacccaccacccaccgccactccccacccagtgctgactgaggagagactgccctgtctctgttgtgttctggtctgcttctagctgactgggtcagcttctgggtatgcatgcagtgagccaggattggccatatctcagtgactaacgttggcaatgtgaactgaaggggaacagggtgctgctgctgttggggtcctgaagagaaagagtctgtgtgtagagagaa
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: TGGTGATCTCTCCCGCAGCA and TTGAGCCTCTAGTCCGCACC. This resulted in a 687 bp. deletion of Chr1:165,655,350-165,656,036 (GRCm38/mm10) and removes exon ENSMUSE00000397738. Founder 11M
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 73396 | ACA GCT AGT CAG GGT CAT C | Wild type Forward | A | |||
| 73397 | GCT TCC GCA CCC ATT C | Wild type Reverse | A | |||
| 73398 | Fluorophore-1 | CAA ACC GGC AGC CCA GAG G | Quencher-1 | WT Probe | ||
| 73401 | Fluorophore-2 | TCA CCA GTG AGA TCC AGA ATG AGA | Quencher-2 | MUT Probe | ||
| 74208 | AGA TAA TGT TCC TTC ATA TCT TCT TC | Mutant Forward | A | |||
| 74209 | TAC AGC ATA AAC CAT CAG GG | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 73396 | 0.40 uM |
| 73397 | 0.40 uM |
| 74208 | 0.40 uM |
| 74209 | 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.