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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
>chr16:84732093-84732195 103bp GGTACCAACTTGTCTGTGTTTGG AAGTTATGTCAGTAGGAGTGGGACT
Mut= 137 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TGATTCTGGACGTATAAGCCGAATAAACCTTTCCCTCCTCAACTGACTTTTTGGTTATGGAGTTTTATGGCAGAAATAGAAGCCTAGACTCAGACAGATATATTTTCCTTTTCTTTTAAATTGTGGGTACATCAATCCATGGATTCACGTTGTTTCTCTTTAAATATTTGCTTATAGCTCTTTGAAGATTGTTTTGTTAATTGTAGATTAAATTCAGACTAGACTTAGCTTTAATAGATAttgttaaaggttttggtaattttgttaaaaatgactttgcatgtggatttttaaaaaaaaattcacatatatatgtatacacacacacacacacatatatatgtcataaatacataatatttctgcaaaagtaatccccagaattttgcttcaattgttctgtttcataatgtagtgcttaagcatgtaatctttcattgtggattttttcacatttctatcataaatcaattttccatccctagatctgagtgagtggtactgcagtaagtccatcctggctcttgtggacggacaaccgtgggacatgtataagcctctgaccaagtcttgtgagatcaaatttcttactttcaaagaccctgacccaaaagaagtgaataaggtattttcctgtctcctccatttttttcttcttctaaatcaacctatgtgcaatggagtatatagtctaaaaatacagacaaactcatatgtggtattctactaaatagtgttttactaggtatttttgtagttcaaaatactggtggtaccaacttgtctgtgtttggattgaccgcatgtcatttgtgctcataactttggataaccctctaaATCATCGGGAGTCCCACTCCTACTGACATAACTTGAATTTGTTCTGAAATCACCGTCTTCAGTGGATGGTTTTGGTAACTTTTTGAGTCTGTCCCACCCTCAACCTCCTGACTTTTACTGCTTAAAACACCACCTTTTATAGTTGTATTAAGTTTTATTACTGACGTTACGGGGTGTCTATATCTTTTATCTCTGAAGAGCCTGCCTTCGAGTTAAAGCTGCCATAGCT
This mutation is a 600 bp deletion beginning at Chromosome 16 position 84,732,127 bp and ending after 84,732,726 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64671 | GGT ACC AAC TTG TCT GTG TTT GG | Wild type Forward | A | |||
| 64672 | AAG TTA TGT CAG TAG GAG TGG GAC T | Common | A | |||
| 64673 | CAT GGA TTC ACG TTG TTT CTC | Mutant Forward | A | |||
| 64674 | Fluorophore-1 | ACC GCA TGT CAT TTG TGC | Quencher-1 | WT Probe | ||
| 64675 | Fluorophore-2 | TTG CTT ATA GCT CTT TGA AGA TTG TTT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64671 | 0.40 uM |
| 64672 | 0.40 uM |
| 64673 | 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.