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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= 129 bp
Wild Type = 150 bp
X-Linked
Sequence
Wt Sequence (deletion in UPPER case):
ttagctaatgtcagtaggagcagtatctgtaggcgagcagtgttcaggctgggagagaaagctatggtggccattttctgcacactgtcaatgtctgcatcttcccttaaccctgcattcattttttaccacttcctgtttaagatttgtccttcccatgttataaacattagagttctttgattaatgcctctgCTAGATTTACCACAGAGCCATTGGCAAATTTATCTTTGTATGACCTGTGATATTTCATAGAATGAGATAAGACTCATGGAAATGCTTAGAAAACTTTTTAAGTTAGAGACAGTGCACAACAAGCATTTCGTAGATGTGTGTTTGAGTTAATGCCTTAGCAGGTACTGTCTTAAACGAATTTCTCTTGCAGTGTATGTCTAATTTGGTTCTTCTGGGTTAATTGAACTGTTCCCTCTCCTAGTGTGTTGATGGAGTTTTGGGTGGTAACGATTATAATGAGAATAACATCAACCAATGGACTGCAAGCATAGTGGAACAGTCTATAACACATTTGGTCAAACTGGGGAAAGCTTACAAGTACATTGGTAAGTGATGACACGCTGTCGTTCCTCCTTAGCTCACGTACCTTTCTCCTTCTGATTCTCTCTGGATTTTAAGAGTGTATCTGAATGATAGGCACTGTAGTTGACCACTCATAGTGGAGGATGCACTTCTGACTCATCGCTTCAACAAAATGCACAACAATGTGAAAAAGGAAGGGCTTTCCATCTGAAGCAGTTTGAACCGCTTGCCCTTTGTAGCAACAGTTGGCTTTGCCTACTGAGACCCTTCagtgctgtgttcctctcggccatcagtgcctctgcataggcctgtgaagatccacacacccagtacaaagtaaaatagtccccaaattctgtctgaaatctgttaatggcacagagcacacaatttaatccagaatttttctgccatgcaattgctggtggtgatagttacagtccatgaaatggctgaatctcatgaagttaagttcaggacggtaggaagtattcaaaataggagacgattttagtctaccagagaaatcgttttgtgttac
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: GAGAGGAACACAGCACTGAA and CTCTGTGGTAAATCTAGCAG. This resulted in a 612 bp. deletion of chrX:9,656,639-9,657,570 (GRCm38/mm10) and removes exon ENSMUSE00001271184
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74557 | CTG TTT AAG ATT TGT CCT TCC C | Common | A | |||
| 74558 | TCT AAG CAT TTC CAT GAG TCT | Wild type Reverse | A | |||
| 74559 | TTT GTA CTG GGT GTG TGG AT | Mutant Reverse | A | |||
| 74560 | Fluorophore-1 | ATT TAC CAC AGA GCC ATT GG | Quencher-1 | WT Probe | ||
| 74561 | Fluorophore-2 | TGT TCC TCT CGG CCA TCA GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74557 | 0.40 uM |
| 74558 | 0.40 uM |
| 74559 | 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.