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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
>chr3:51493701+51493808 108bp CTAAAGAGCCACAAGCCATTG CACACCTATGACTCAGCACCA
Mut = 85 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GGCCCAGAGGTCAGAGGTACTTGATTTCTGCATCCCACAGGTCAGTGGACAACCAACTCCTGAGCTGATCTCTGACCTGCATGCATTCAGCATGGCGTGTGTGCACCTGTGTGCACACGGGATAAAAAAACTAAAACTGCTTTTAATCCCAGTACATTTGACTCAGCATGGGCCTCTCATAGCACTAGGCCATAGGTATTCTAAATGGTTTTTGTGTGTTGTATCCTTTTCCCGCAGTGATTTTTAAAAACATTTTAAAAGCCCATACCTGTTCAGATTTATAATCTGTATTTAGTAGTCACATTGTGACTAGACTAGGATTATGAAGCGTGGACCCTGCAGGTAAAGACGGGCCAGAGAGCGGAGAGGTGTTACGGGCAGGAACAGTGGCTGAGTTGCTCTGTTTGTGCCATTGGACTGAGCTTGTCGACTTTGCTTTCTGTTTTCCTCTCCagatccagttgtgggacacggcagggcaggagcggttcaggaagagcatggtccagcactactacaggaatgtgcatgctgtcgtctttgtgtatgacatgaccaacatggccagcttccacagcctgccggcttggatagaggaatgcaaacagcacttgctggcgaacgacatacctcgaattctggtgggaaataaatgtgacttgagaagcgccattcaggtgcccacagacttggcacagaagtttgctgacacacacagtatgcctttgtttgagacctctgctaaaaaccccaatgataatgaccacgtagaagctatattcatgaccttagctcataagctaaagagccacaagccattgatgcttagccagctgcctgataacagaattagcctgaaacctgagaccaagcctgcggtgACTTGCTGGTGCTGAGTCATAGGTGTGGCCATTGCGGGTCTGACTTACCTGAAGAGATGATTGGGAAGTGTGACAGTGATGAAGTTCGTAAGAGTAAATCCTAACTGTAGTAGATCATTCTGCTGTTATCGTTGTTATGATGCCTTTTGTATTTTGCATCCTCTCAATCAAGTTTGTCACTGAGACAGCACATGGAAAAGGCCGCTTTACAGTGGGGATTGAAAATGAAGACTACCGGGGCCATCTTTCCAGCTCCAGCCAGAAGCAGCAGCTTCAAGTGAGCATGATGGACCTTAAATGGTTGGTCCTCTTCCTCTCTGGGTCCCTGGAGCAGCAGAAGCCATGCTGGGTAGTGC
This mutation is a 428 bp deletion beginning at Chromosome 3 position 51,493,354 bp and ending after 51,493,781 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48622 | CAC ACC TAT GAC TCA GCA CCA | Common | A | |||
| 48623 | CTA AAG AGC CAC AAG CCA TTG | Wild type Forward | A | |||
| 48624 | TTG CTC TGT TTG TGC CAT TG | Mutant Forward | A | |||
| 48625 | Fluorophore-1 | CAG AAT TAG CCT GAA ACC TGA GAC | Quencher-1 | WT Probe | ||
| 48626 | Fluorophore-2 | TGC TTT CTG TTT TCC TCT CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48622 | 0.40 uM |
| 48623 | 0.40 uM |
| 48624 | 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.