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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
>chr1:14910565-14910688 124bp AGGCCAGAATCCACATCTGA CGTCACTACCACCACCATCA
Mut= 111 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GTATCTTTTGGGCAATTGCAAGCTAATTATAAATATAAGTCACATGGAACCTAATTATGATTTGCTATAATGTAATCTCAGCATTATAGAACAATTATGAAATTAATATGCAGATTAAAAGTATAGTAACAAGAGGATCTGTTTCTATAAATACTATGTAAAAAATTAACCCAGTGTTTGGATCTATAGGTACTTTCACCTGGTCTCAAGTGGTGTCCATTGGgtaattctgaagctaaaactacatcctatttctatagttcaagaaagtgtacctggttttataagcacttgactccgaggttcggagtgggtagttcttcaggatatattaagtaaatcccaaacaacatttaatgcatattttgttatttctttagtgctgaatataatggatggttatggaaataccccactgcattgtgctgcagaaaaaaatcaagttgaaagtgtaaagtttcttctcagccaaggagcaaatccaaacctccgaaatagaaacatgatgtcaccccttcacatagctgtgcatggcatgtacaacgaagtgatcaaggtgagcctgctggccatcacagtgaggctgactgtggagctaggccagaatccacatctgactcacttactgtttcaaagtgactgacttcaagtgccatgtcatggCAAATGAAACGTAACTATTATGTTTATTTTTCGCCAAATGATGGTGGTGGTAGTGACGATATGACGGCAATGGGGAAGATGATGATGGATAGCACTGATGAGAAAGATGGTGGTAACGACTGTCATAGTAACCAGAGTTAACTTTTCTTGAGTTTTCCTTTATCAGGTATTTTAAAGAAGAGCTGTGTGACTTTATTAAGTTACCATTT
This mutation is a 440 bp deletion beginning at Chromosome 1 position 14,910,623 bp and ending after 14,911,062 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40826 | AGG CCA GAA TCC ACA TCT GA | Wild type Forward | A | |||
| 40827 | CGT CAC TAC CAC CAC CAT CA | Common | A | |||
| 40828 | CCA GTG TTT GGA TCT ATA GGT ACT TTC | Mutant Forward | A | |||
| 40829 | Fluorophore-1 | CTG TTT CAA AGT GAC TGA CTT CAA GT | Quencher-1 | WT Probe | ||
| 40830 | Fluorophore-2 | CCA TTG GCA AAT GAA ACG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40826 | 0.40 uM |
| 40827 | 0.40 uM |
| 40828 | 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.