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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
>chr5:129854713+129854806 94bp GAGAGAAACTGGAGCTTCC TCCTCTTCTGTGGGCAAG
Mut= 87 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
CCCCGCCAGGAATGATGAGTCAGGACATTTTAGGAGTAACCCCGTTTCTTCTGCTGTAACTCTTTTCCATATTTCCCACTGCTCTCTTTACTGAAGAATCCATCTTTTTGTCTCTCCCCTCCTCCCTCCTGTCTCACGGCATCCCACTCTGTAGCCCAGGTTCCTCCTTCCTCAACCTTCCGAGTGCTGGAAATCCAGGTGTGTGCAACCGCTTGTCTCTTCCGTTTCCCCCTCACCGAGCTATCCTTCCCGACCTCCCTCCTctacctaatataaaagctgaactgcgtcaccccgagccaagccaggttttttttttttttttttcagcctttccctagatcccagaagcctgtctcacttcaccctgtctgtcagcctgcaggtcccggctctggcatccgagagaaactggagcttcccgtggtacacgtgagctggaacgacgctggtgcttactgcgcatggcgggggagacgcttgcccacagaagaggagtgggagtttgcagcccgagggggcttgaagggtacgtgggtcatcttcccgccctgtcctgcactggcttgaaaggatggggctggtccgaatcccgccccGAAGGGATTTTCGAGTCCCCTCTCGTAAGGATTTTTTTTTTTTTTTTTTTTGAGACAGGTTTTCGTTTCTCTGTATAGCCCTGGCTGTCCTGGAACTCACTCGGTAGACCAGACTGGCTTCTAACTCAAGAAGCTCCGCCTGCCTCTGTCTCCTGAGCGCTGGGATAAAGTAATCTCTGCCCAGCTAGAGTAGTGATTTTAACGTGGGTGACAGTGTAGGATAAAAAGACAAAGAAATCCCACCAATCCCTGAGCTCACTCTAGCTCAGGATCTGAAAGCTCACCAC
This mutation is a 336 bp deletion of Chr5:129,854,573-129,854,908 (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 71586 | AAA ATC CTT ACG AGA GGG GA | Mutant Reverse | A | |||
| 73347 | GAG AGA AAC TGG AGC TTC C | Wild type Forward | A | |||
| 73348 | TCC TCT TCT GTG GGC AAG | Wild type Reverse | A | |||
| 73349 | Fluorophore-1 | TGG TAC ACG TGA GCT GGA AC | Quencher-1 | WT Probe | ||
| 73350 | CTT GTC TCT TCC GTT TCC C | Mutant Forward | A | |||
| 73351 | Fluorophore-2 | CCT CCT GAA GGG ATT TTC GAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 71586 | 0.40 uM |
| 73347 | 0.40 uM |
| 73348 | 0.40 uM |
| 73350 | 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.