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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
>chr16:30356912-30357023 112bp GTCTCGTCTCCATCGTACCC ACATGCTTCCTCTCAGAACACA
Mut= 109 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AAATAGGAGTCTAAAGGAAGTAAACCGCTAAAGTTTGATTACCACTATTGATATCTTTGATTTAAATAGTGTAGCTAGCTAGCAATTTTATTTTACTTCATTTATTTATTTTGGTTTTTGAGATAGGATTTCTCTGTGTATCCTTGGCTGTCCTAGAACTCAGTCTGTAGACACAGTTGGCCTCGAACTCAGATCCAGCTGCCTCTCTCTCCTGAGTGCTGAGATTAAATGTGTGAGCCGCCATGACCTAGCAGCTTTTTTATTTTTAAAAGTAGAGAATTTTATAGTCATGgaataagacttggaagcatgaatctcagaatggttgactttagccatatctaacataattcttcttctaaattattaatataattactgcatttttctgtcatagaaagttgatttatggagtaaatgaaattgctgtgaaagtgccttctgtttttaagcttctaattaaagaggtaagactttgtatataacccaacaaacagtagagtcatccatttggaaagagtcatgtttaagaagctgttcttagtttcctgctttgcttcatattttacttggaaggtaaaaattgaaaaattcttgtctagtagaaaaggtatgagtatgcattttttattatatagatttctttgtgtctcgtctccatcgtaccctggttaagtgtttgtgacttgctcccttgctcctttacttcacattgGCTAGTACTGGAAACTGAACATTTGTGTTCTGAGAGGAAGCATGTTAGTTCTTGCAGTGAGGATTCATTTATAATTGCCTGGGCTTTCTGAATAAAAGATTAGGAATGTGTATGGGAAGTTGGTGAGTAACTGCCCAGCAGAAAGACTTGCCAGTCTTTCTTTGGGTTTTGGCTATAGATAAAAAGTAGTATTGGGCGGATACTTTACTAGAAACAATGATGTTTTCCCACCACTTTCTTAGAGCAGCCCTTTGGTGCTGCCTGAGAGAATAGTTTCAGGTATGATTAAGACA
This mutation is a 423 bp deletion beginning at Chromosome 16 position 30,356,957 bp and ending after 30,357,379 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48044 | GTC TCG TCT CCA TCG TAC CC | Wild type Forward | A | |||
| 48045 | ACA TGC TTC CTC TCA GAA CAC A | Common | A | |||
| 48046 | ATG TGT GAG CCG CCA TGA | Mutant Forward | A | |||
| 48047 | Fluorophore-1 | ACT TGC TCC CTT GCT CCT TTA | Quencher-1 | WT Probe | ||
| 48048 | Fluorophore-2 | AGA ATT TTA TAG TCA TGG CTA GTA CTG GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48044 | 0.40 uM |
| 48045 | 0.40 uM |
| 48046 | 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.