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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= 73 bp
Wild Type = 80bp
>chr13:106948663+106948782 120bp AGTGGGCCAGAGCATAAAGG TGGCCTTTTCTAACAGCTTGA
Sequence
Wt Sequence (deletion in lower case):
ACGTTTTGTATGGTTACTATATTCTTAAAACTGTACAGTGGTTAAGAATTGAGAAATCTTTTCCATTTGAATGCACTCTTTCCCATTTGAACTCTTTGGAAGTTACAATTAGTTACAGTTTGCTGGAATTATTGCCAATGTAGAAAGCAGGAACCcaccagtagaaccgattgctcttgcttttgttctggctccagaagctgagtatatcccctaagttaggaaatacaggttttcacttatacctaatgcctttgaggtactactgctgttaaccacataaaccctgaaaaaaatgaagccagagtaaaatgtctttaactgtacattgataaacagaaacagtcacttacagactttgtgttcaagtgggccagagcataaaggagttgtgtttatttataggctgctttaagacctaccgatgtggtgctggaagttgggcctgggactggcaatatgactgtcaagctgttagaaaaggccaaaaaggtaaaagtggggtttcgtgtgttactctcaaattagctcagctggtttacaaatgtgaatgttttaataaattggcattgttccaatagtagttagtgggcccttagaaattttatctctaaaaatgagatctttctatcaaagaaaacaaaatctacacttgatgtattggtgggtgatctaatataggaatgggccatgtgtttaaaggagtaataataaaatagaaagtctgagataacccacagggcagcagctaaccattatgaagcctggaagaacagagccgtgaagttaaagtcattaggaccaagttcagcaggagactcagagggtaggatgatgatgaagaatgggtcagggagtcaaccagaatcagaaactcaaatggctacgatgtagaagtggtcacgacacttggtgaagcccctgtggatgtggcaggaaaatagttcagctttatccacttccattcacctggagtgctatggtttctactctggatgtcagttgatgagagtacttgtaggtcccttctatgctgtctaaaaaccactaactacataaagtgttgtcatcatctgttcagtaacacagcatggagacagatcagacaggatgttgttttaagtgtagaaaagagtagagctggtgtttgtgttaagactgaaaaaagcttaagtcattgtagctcctatgagttattactgggagggtaaaaaacatttatactccacaatcaagcttttttgttattgttaatctaggtagttgcctgtgaacttgatccaaggctagtagctgaacttcacaaaagagttcagggcacgtgagtatatgtaataactaaagggcatttcagtcttctcaaacaagctcttacaatttctctccttgttttcaggcctctggccagcaaactccaggtcctggtgggagatgtactgaaatcagatttgccattctttgatgcttgtgtggcaaacttgccttatcaggtatttctcaagctgtcagaagcgtcacactgagcatttctctggtgtgtccttcctcagagctaactgtaattctctttcagatctcctctccctttgtcttcaagttgctgctccaccggccatttttcaggtttgtgcaaaacagtccctgttaagctttagacaattctggcaagaaacacaggggggaaaaaaaacCACAACAAGAAACTGCTGGAGAACTTTGTAGAAAGTAACAGTTTCTGGGACAGGAGTGTGGTAGCTCAGTTGTTGAAGTGGCGTGCTTATTAGTATCCAGAAAGCCCCTAGGTCAGTCTCCAGCAGCACAGAAAACTCAGCATGGTGGTGAG
This mutation is a 1536 bp deletion beginning at Chromosome 13 position 106,948,451 bp and ending after 106,949,986 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68753 | GCC AAT GTA GAA AGC AGG AA | Common | A | |||
| 68754 | GGG ATA TAC TCA GCT TCT GG | Wild type Reverse | A | |||
| 68755 | GTC CCA GAA ACT GTT ACT TTC | Mutant Reverse | A | |||
| 68756 | Fluorophore-1 | AGT AGA ACC GAT TGC TCT TG | Quencher-1 | WT Probe | ||
| 68757 | Fluorophore-2 | CCC ACA ACA AGA AAC TGC TGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68753 | 0.40 uM |
| 68754 | 0.40 uM |
| 68755 | 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.