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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
>chr6:17834879+17834966 88bp AGCTGTACCAGAGAAGTATCACG GATAAAGCTAATGGGAACGAAAG
Mut= 83 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTTCAGCTAAGATGTTTTTGTTATTTGAGTATTATATTAACTTTGAGAAAAATAATTTGGAATGCACCTTAACAATATTCTTTTATCTATCACTATATTTATTGATTCTTTTAAAAACCACTCTTCCAGCAGAGAAAAACCATTTTAActaaataatattggtatttagttactaaaatctattacagttagaccaatttaaacatttttcaaaggcggatagaaaaagtaaacatatattaagggaatgtatcctgaaattacaagtagaaactgaggtcataaacacacattatctttctgttctagatatttgaatggtggtattttcgcaagtatggaacatctttcattgaacaagtctccgtaagccacttgcgcccccttctgggaggggttgacaacaactcttccaataattctaattccagtaatggagactcagattccaacagacaaagtgtctcaggtatggaacgtctcttacaaaattttcgtgaaattaacaaaactgttttaaaagattacgtttcagagaaaaactgtgtttaaatttcaagggaggtaagaaagctgtaccagagaagtatcacgcatgcatcccagggacagaaaCTATTCATGCTGTTAACTTTACTTTCGTTCCCATTAGCTTTATCATAACAAATGATAATAGCATTAGTTTTAAAGCAAATGTAGCATAGAGTATAGAGTATTACATAGAGTATCAAGTGCATTAGCTACCT
This mutation is a 467 bp deletion beginning at Chromosome 6 position 17,834,456 bp and ending after 17,834,922 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40946 | AGC TGT ACC AGA GAA GTA TCA CG | Wild type Forward | A | |||
| 40947 | GAT AAA GCT AAT GGG AAC GAA AG | Common | A | |||
| 40948 | TTT AAA AAC CAC TCT TCC AGC AG | Mutant Forward | A | |||
| 40949 | Fluorophore-1 | ATG CAT CCC AGG GAC AGA | Quencher-1 | WT Probe | ||
| 40950 | Fluorophore-2 | AAC CAT TTT AAC TAT TCA TGC TGT TAA CT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40946 | 0.40 uM |
| 40947 | 0.40 uM |
| 40948 | 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.