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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= 105bp
Wild Type = 112bp
Sequence
Wt Sequence (deletion in UPPERCASE):
ccttggtgggtcgctacgggagccggcgtctttcttgtctggcccgaccctgctctagccatctccgctttcccattcggccatccccgctaggctctcagaagtcaagcccgcttctcttcttccgaggacttactgttttttgttttttgttttttctgtttgtttcaggagctctgccGTCCGTTTATAAGAGAGACGCCGACTCAGGTGACTCTCAGAATCCTCCGAATCAGCCTTCCAAACAGTCCTCCACTCCCCTGCCTTCTTCTAACCAGGTAAAGACTACAAGGCCGACCGACGGTCAGGGGCAGAAGTCAGATAAAAAAGACCAGGACAAGACGACTTTGGCAGCAGTTTCCAGTAAGGCGGAATCTGGGCCGCGGACCGCCGCGACCGATCACAGTTTGGGTGATTCCAGAAGGCAGCCCGAGAAAACGGATGCCGAGTTGAAcGAAACTGCGCGGCCCCTcTCACCAGTCAACCCCAAACTGGAAAAGTCTGatcagagctccacagaagattcaGGCAAGCCCACAGGAGGTAATTCTGGCAAGCCCACAGGAGGTGATTCTGGCAAGCCCACAGAAGCTGGTTCGAATAAGGCCACAGAAGATGATTCTGGCAAGTCCACGAAAGTTGATTTGGACAAGCCCACCTCCAAAATCTCCCCAGATACGGAGACTTCGAAGACTGACAAAGTCCAGCCAACTGAAAAAGGACAAAAACCAACACTTACTTCCAAAACTGAATCTGGGGAAACACTGGCAGGGGACTCTGACTTCTCTTTAAAGCCAGAGAAAGGAGATAAGTCTTCAGAGCCTACTGAAGACGTGGAAACCAAGGAGATTGAAGAGGGTGACACAGAGCCGGAAGAAGGCTCACCACTTGAAGAAGAGAATGAAAAGGTGCCGGGCCCTTCCTCCAGTGAGAACCAAGAGGGGACACTTACAGACTCTATGAAGAATGAGAAGGATGATCTTTATAAGGACAGTTCTGGAAACACCAGTGCAGAGAGCAGCCACTTCTTTGCATATCTAGTGACCGCTGCTGTTCTTGTTGCTGTCCTCTATATTGCTTACCACAACAAACGAAAGgtaagaggggactgggatttccaggaccctaagttctttgttcatggacagctatctgtcactcatctgcctgtgagctttgagaattttaagatcatcttgttttttccatagaccctgctggggcttataaaactaagttttgtaaatcaggtgttttcatcatgtgacttgtttcctctcagttctgctgtaaagtgcca
This mutation is a 916 bp deletion beginning at Chromosome 6 position 72,615,524 bp and ending after 72,616,439 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68845 | AGC CAC TTC TTT GCA TAT CT | Wild type Forward | A | |||
| 68846 | ACT TAG GGT CCT GGA AAT CC | Common | A | |||
| 68847 | CCG CTT CTC TTC TTC CGA | Mutant Forward | A | |||
| 68848 | Fluorophore-1 | TGC TTA CCA CAA CAA ACG AA | Quencher-1 | WT Probe | ||
| 68849 | Fluorophore-2 | CAG GAG CTC TGC CGT AAG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68845 | 0.40 uM |
| 68846 | 0.40 uM |
| 68847 | 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.