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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= 74 bp
Wild Type = 106 bp
>chrX:153130686-153130791 106bp ATAAGGGAAATGTTGTCGAG TCCACTCAACTACAGCCAT
X-Linked
Sequence
Sequence (deletion in UPPER case):
tgcatgcacagacaggggatcctgagagcaagctggctagtgagactcacagtatctatgagctctgggtttcactgaaagaccctgcctcgaagaatgaggtggaagagcaatggagggaaaaatcttaatgtcaacttgagcctccacacacatgtgcacatgtatgcccacatacatacaagcatgcatacacacacttacacataccacacatatataagaaaagaagaaaacattaaagatgaaagaggaggtctatcagctctggttggcgcatgctctgtttgatgctctccCTTCGAGCATAGGCCAAGACACGGGTGTCCTCCTGAAAGAATCGGTTTCCCTCATCAGTGAGCTTCCAAAGGAAAGAGCGAGGTTTTCCATCTGGCTCATCTTCAAGGTCGACTGGCACCTTTTCAAAGCAGGTCAGGGAGCAGAGGTTGTGGCGAATGGTGTTTTTCCAGCCCTCTGGTGCTGTCCGAAAATAAGGGAAATGTTGTCGAGTAAAATTGTAGATCTGTTGCACATTGAGGCCACAGGAAGGGCTGCTTTTTAATGCTAGAGCAACTAGATGGCTGTAGTTGAGTGGAGGGCGTGGCCAGGACTCCTTCTCCCGGTGGGGGCCATGGCACACTGTGTGTTTCTTGGAGTATTTCTTGGAGGATGTACATTCCTGTTCCTTAGTCTCCTCTTCTATGAAATCAGGGGCAGTGGAGTAAATGAGCAGCTTTTGTTGCTGATGATGCTCAGTGTGGAGAACAGACAGGGATTGTACCATTCTCTGGGTCCCTAAGTAGTCAGACTCGCCAGTCTGTGGGAATGGTGCACTTGGCAGAATTTTATGAATGGTATTTGGAGCCTCCTTGCTGTTGATGGGGCACACAATGTTGGGATTCACCCACATCCACAAGCTGGGTTCAGCGGGAGGACCATCATCATCAAAATTGGATTTCTTCTTCTGAGGCAGTGCAGGTGGTTTTGTCAGTTGGATTTTATATTTGGCGAGACTCTGCTCAGCGAAAGAGCACTGATCCATGGTACAAGGCAGGAAAAATTCATTCCCCATGTCCCAATCCAGCATCCCAGGGACTTGGCCATGGAGACTGTACCAGAAGTCCcgattttttacctttacatccatctttaccgaagtgatggatgaaggtaaacttccaaatagataaaagtctctggaaatttgccaagctattgctgaggcactatttaatgcaaaatgtctcctctccagtgcaatctatcagtaggcctgctggaaaagctgacacgaggacactgatacttcactggtcagagaagccacagctttctggtacacagctcaaaagagtaactaagacgatcattttattaggaaaaactgctttcaaatttttccaagtcttttcttactgttgctcacaaatgatgttttga
This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences: AGGTAAAAAATCGGGACTTC and TGTCTTGGCCTATGCTCGAA. This resulted in a 825 bp deletion of ChrX:153,130,158-153,130,982(GRCm38/mm10) that removes exon ENSMUSE00000621768. Founder 07F
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 73384 | ATA AGG GAA ATG TTG TCG AG | Wild type Forward | A | |||
| 73385 | TCC ACT CAA CTA CAG CCA T | Wild type Reverse | A | |||
| 73386 | Fluorophore-1 | AGA TCT GTT GCA CAT TGA GG | Quencher-1 | WT Probe | ||
| 73387 | ATG CTC TGT TTG ATG CTC TC | Mutant Forward | A | |||
| 73388 | AGT TTA CCT TCA TCC ATC AC | Mutant Reverse | A | |||
| 73389 | Fluorophore-2 | TTT ACC TTT ACA TCC ATC TTT ACC GAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 73384 | 0.40 uM |
| 73385 | 0.40 uM |
| 73387 | 0.40 uM |
| 73388 | 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.