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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
>chr5:23776133-23776223 91bp CCCTAAATGTTGATGGAC TGGAGCACATGCACTTCTATAC
Mut= 90 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCAAAACCTGGGACTCACCAGTGCTTATATAGACTGGCAGGCCTGAGGCGCCAGGATCCTCTCCTCTCTGCCTCCCCAGCATAACCCAGGCCTGTGTTACCACACCTGACTTTTTCACCTCAAACGCAAGTCCTCATCCCTGCTTGCTCCTCAAGCACTGTACTGACGGGGCCCTAAATGTTGATGGACTTACTTTTGCGTTGGTTGatatgcgctatggtaatattttagaagctcctagtatagaagtgcatgtgctccattaaaataagagaagattgcttggtattatttgccaataaagattttccttaaatctcttgttttgaacaatggtgaactttgtttactttttactaatagtaaaattaggtagaaagtaaaacaacatccacttcttgaaagctttggttttcttttcatcagatactctgactttgtcgttcacgaaatcgggaaagatggacggatcagccatttggatgacttatctgttccggtggatgaagaggtaaaaatgtctctagatcaagctctcttctgtatgtagtcactcttcctaatgatcctgGACAGGACGTGTTACTGTCTGTGTGTGACTCTCTTGCTTTCTTGTGGTTGAAGGACCCTCCAGAAGACGCTCTCACTGTCTTGACAGCTGAGGACAGGCAGCAGTTGGAGGAGCTCCAGCTTTTCAAAAATAAAGAAACCAGTGTTGCCATCGAGGTAAGCGGCGCAGGAAGTGACGTCTCCGGGCTGGGCTGCTTCTCATTGGGTG
This mutation is a 364 bp deletion beginning at Chromosome 5 position 23,775,824 bp and ending after 23,776,187 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47706 | CCC TAA ATG TTG ATG GAC | Common | A | |||
| 47707 | TGG AGC ACA TGC ACT TCT ATA C | Wild type Reverse | A | |||
| 47708 | CCT TCA ACC ACA AGA AAG CA | Mutant Reverse | A | |||
| 47709 | Fluorophore-1 | ATG CGC TAT GGT AAT ATT TTA GAA GC | Quencher-1 | WT Probe | ||
| 47710 | Fluorophore-2 | TGG TTG GAC AGG ACG TGT TAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47706 | 0.40 uM |
| 47707 | 0.40 uM |
| 47708 | 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.