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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
>chr3:30800786+30800909 124bp TACCGTTGAGGGAAGAAGGA CTGATAAGCATCTTCACAGCAAG
Mut= 122 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTAATCTCTAAATCGAATGTATTTCTTTGCTAGTATAGCAGTAGGTTGTTCAGAGTGATTCTTTAGTGGTTATAATAACACGAGCATTTTCATTACCGTTGAGGGAAGAAGGAAGTGTTTGAGGTTATTTTTTTGTTGATTATTCAgagattgatggttaacaaatgtttatcttgcttgaataataatatttacttgctgtgaagatgcttatcagtatactcacagttgacataaataacattatagtcaatagacttgatattctgttttattttctgttgtcagaatgttgacatttatagcatgatttcataagacgtaagttcatgaatactaaattttaaataaccaatttcttgtatagcttcaaaagcagtggagtgcctcttggattcaaagtgggccaaggccaagaaaggagaggacgctttatttacaaccagggagtctgtggtcgactactgcaacaggtagcgttccttggtgaaggataacaattagcaataccagttttttccatagtgttgaaactgtttaactaaatgtaatgagctttatatgtaacttggatttttcagtagaaatatctttgttcttaaacttactatatttttagttgtgtatgtatactttgtagttatgttcttttacttgtgtgtatttaccagagctactgaatgGAAGAATCAAGATTTTAAAAAGGATAAACATGTCTACATTGAACAAATTCTGTGTAGGAAATGGTCAGGATTTTTTTTCCAGATCTTTTATAGGATGACGGTTAACTTCATTATCCTTTGGTTTTTAATGGCTTTGCCCTGGGCTATAAGCCTAACTTACTTGCTGTTTT
This mutation is a 537 bp deletion beginning at Chromosome 3 position 30,800,839 bp and ending after 30,801,375 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41242 | TAC CGT TGA GGG AAG AAG GA | Common | A | |||
| 41243 | CTG ATA AGC ATC TTC ACA GCA AG | Wild type Reverse | A | |||
| 41244 | CCT GAC CAT TTC CTA CAC AGA | Mutant Reverse | A | |||
| 41245 | Fluorophore-1 | TGA TGG TTA ACA AAT GTT TAT CTT GC | Quencher-1 | WT Probe | ||
| 41246 | Fluorophore-2 | TTG TTG ATT ATT CAG AAG AAT CAA GAT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41242 | 0.40 uM |
| 41243 | 0.40 uM |
| 41244 | 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.