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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
>chr15:81768560+81768667 108bp GCCCTGAGTGGGTTGTTAGT GGACTTCCATTTTCCCTGCT
Mut= 111 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCACGCTGGGCTTACCCTGGCCCTTTTCATCTTCTGCCCCCTTGAGCATCAGTTTTCCCCTCCACGTGGGACCAGGAGACAAGCCCTGCCCTTCTCCCAGGCTGACACAGTGCAAGCTCTCCTGAGCCAATGCCCTCCTCCAAAGGGAGCTGTAGTCTTACCCACAAGAGAGAATGTGTAGCCATTGGCCCTGAGTGGGTTGTTAGTGTCTGGTTGAGGGGTTGGCTGCTGTCACTGGGCctgccgctgactaactgagcagactcctctgggagagcagggaaaatggaagtccttgcatcagaactgagtgactccatgctgtccggtcacctcttacttcagccacagctgtctgaggcctactcttggtccacctcagccctgaagtgccagcctcagggctcctcatgggaccaacctgggttcaccctgtcctgtgtctctcttttcccactaggtcaacactacccctgaagcaagaggagtatgaggtgagtgtcgctgtggtgggtggatgcggtgagggCTGCTGTGCTCAGGGCAGAGGGCTGGGACTGAGCCAGGCTGGATGGAGACCCGCTTAGTCCCCTCGGACCTGCCTTGCGCAGGCTTTCCTGCTCAAGCTTGTGCAGAACCTGTTTGCCGAGGGCAACGACCTGTTCCGGGAGAAGGACTACAAGCAGGCTCTGGTGCAGTACATGGAAGGGCTGAACGTGGCTGACTACGCCGCCTCTGACCAGGTGGCTCTACCCCGGGAGCTGCTGTGCAAACTGCATGTCAACAGGGCAGCCT
This mutation is a 289 bp deletion beginning at Chromosome 15 position 81,768,613 bp and ending after 81,768,901 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48006 | GCC CTG AGT GGG TTG TTA GT | Common | A | |||
| 48007 | GGA CTT CCA TTT TCC CTG CT | Wild type Reverse | A | |||
| 48008 | CTA AGC GGG TCT CCA TCC AG | Mutant Reverse | A | |||
| 48009 | Fluorophore-1 | CCG CTG ACT AAC TGA GCA GAC | Quencher-1 | WT Probe | ||
| 48010 | Fluorophore-2 | ACT GGG CCT GCT GTG CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48006 | 0.40 uM |
| 48007 | 0.40 uM |
| 48008 | 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.