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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
>chr19:8935165+8935274 110bp TCTCAGCTTTTGTTTCTCCATC GGTTTGACCCTCAACACTGC
Mut= 108 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TCTTTCTTTTATTCTTTCTTTCTTTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCTTTCCTTCCCCCCCCACCTCTCTTTCAACTAAAAGTCCCCATCTCTCTAGAGCCCTAACAGTGACCCCACattagcaggctgactgggaggaacctcctgtcagtatctttttttgttttgttttgttttttgtttttggttcgagacagggtttttctgtgtagccctggctgtcctgtaactcacttggtagactcagaaatccgcctgcttctgcctcccgagtgttgggattaaaggcatgcgccaccatgcccggctcctgtcagtatcttgtccccacttctctcaggtcttgacacttgatcctggtcttatacttttacctcttctcaatagccgggggaacaaagaccctctgtccagtcctgggggtcccggatctcggaggagcaattataacttaggtatgtaacagagaagtggagggaagaactcgcatgatggaattagtcaggtacggtgggtttgctgctctggccttcaggagggaggcttatttccctgctttttcctgtagaaggcatttcagtgaagatgttccttcgtggccgtcccatcaccatgtacattccgtctggcatccgaagtcttgaggagctgcccagcggcccgcccccagagaccctgagcctcgactgggtgtatcctattcagttccgtgggaatcacccctgcccacaatgggtccctttccttctttacagtttctttctgtgtgggggtggacaaggaatcctgtgaattatcccataccaccctaatgtggatggtgatgccaagtgggagctttgccgttttcatgttcccttcttcctcttcctgtccttcatctggccctttccttgaccatgcccctcaacacagttatgggtacaggggtcgtgattctcgctctaatctatttgtgctgcgctctggggaggtggtctactttattgcctgtgtagtggtgctgtaccggcctgggggaggcccagggggtcctggaggtggcggccagagacattaccggggtcacacagactgcgttcgatggtgagggtcttggggtctggggctggtgggcttgacaggaagagggtctgtctacggggggaagggtagcaaagacagctgccttgctgcttcttgggtggtcttgagagagacccctttcctatagttctcttccccttccttagccttgctgttcaccctgatggtgttcgtgtagcctcaggacagacagctggagtggacaaggatggaaaggtaatgtcaagcagaatatcgagcagataggatgagataacaggtcagcttttcttcccggttctgaccccttctcttccactagcccctgcagcccgtggttcatatctgggactcagagacactcctaaaactgcaggagattggactgggtgcctttgagcgaggtgtgggggccttggccttttcagcagcggtgagctgagcacaaagcttccgatcttgtttgtttgtttgttgttgttttgttttttgggacagggttcctctatgtgtagccttagctgtcctggggctcagtttgtagaccaggttggctttgttttgttttgttttttcgagacagggtttctctgtgcagccctggctgtcctggaactctctgtggaccaggctggcctggaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaagatgtgcgccaccacatccgactgtttgtttttgaggcagggtctcatgtaccttcggctggcctctgtaactcaatatgtagatgaggatgtttttgaactcttgctccttctgtctctgcccaccaattgttataggcacgtgccaccatatctggctagacccttattcttgctcaggacaccactctttgattttaccaggaagcctggggaaggtctagtgttctgggctcacacagccagttctcctgagaaagagaccctcccaacctgtgatagatccctgtctcatagcaggcatgtacagacgtcccggcttcacatgctgtgtctcgtcctcaggatcaaggtgcttttctgtgtgtggtggatgattccaatgagcacatgctgtcagtgtgggactgtagccggggagtgaagctggctgagattaaggtgaggaacctaggtggcttgatggtttctggatctggggtagggtgggagtggtggaatacagacagttccaggtttacttatacatacacttccgactcagacaaactctcagttctctcagcttttgtttctccatctgtgcattgggaactacaggagttctaacGGGGACTATAATTATTTAGGGGATCAGCTCCCCCCCCCTGCAGTGTTGAGGGTCAAACCAGGGCCTTTAATATGTCAGTAGGCATTCTACCATTTGCTCTATACCTTCAGCTTTCAACCAGAGCCTTGAAGGAAGAATTATCTTGAGGTCTCCGGGCTTTTGTAGACCAGCTGTTAGGAAGATTGAGTTATAGAGTAGACTTTGGTGGCATAAGGCTATCATGCCA
This mutation is a 2241 bp deletion beginning at Chromosome 19 position 8,932,972 bp and ending after 8,935,212 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64395 | TCT CAG CTT TTG TTT CTC CAT C | Wild type Forward | A | |||
| 64396 | GGT TTG ACC CTC AAC ACT GC | Common | A | |||
| 64397 | TCA ACT AAA AGT CCC CAT CTC TC | Mutant Forward | A | |||
| 64398 | Fluorophore-1 | CAT TGG GAA CTA CAG GAG TTC TAA C | Quencher-1 | WT Probe | ||
| 64399 | Fluorophore-2 | AGC CCT AAC AGT GAC CCC AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64395 | 0.40 uM |
| 64396 | 0.40 uM |
| 64397 | 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.