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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
>chr14:41062722-41062847 126bp GGGTGAGTGCGTGTGTATTTC TTGTAAGAAAGGGTGCATGGT
Mut= 110 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCATCAGCAGAATGTCATGATTCAGATCTTAAAGATTCCCCCAAATGGTCACATGCTAAAGGCTTGTTTGTGGATGTGGCAATACTGGGAAATGACAAGAGTCCATAAGACTCCCCAGGAGCCTAGTGGAAGGAAGTAATGTCACTGGGTCACCATGCAGGAGCTCCCACCCCTCTCTTTTTCTGTGTGTGTGTGAGTGGGTGGGTGTGGGTGAGTGCGTGTGTATTTCGTGGGAGGGgttaagaatgtatgtccctcccttcttttttcctccttctttccttccattcttttcctaaagcgagttacttttaccatgcaccctttcttacaatgtttggcctcactataagccaagaatgatggaccaaaggaccataaagccaagcctttccctcttcagttgaagagatcaagcatttttgtcacagtaagggtgatccagaaatagttcttattcttcctggaacgaatctgacttgttagaatggatctactttgaaggttagattgcgcttgtataatagagcatttgatttggtgtgtcctaggttctcagcatggatactgcctacctgaagaactgctttggaaccggcctgacccaggcactggcagaggtggcaagagtgcggcccagtgacccaatagagtacctggctcactggctttaccattataggagtataactgtagctgaggaaaaggtgtgttgtgtcctgggcctgcttaggtagggtgcagcttgctcagaacaacttgaaatccaagttatagtcctgagttggccaggagagtttttggctcatctcccttctgtggattgctttgtcccagatgtctctgaagtatgcaggactcgggggggtaaggaggaaatctagcttaatgggttgtagaattgggttggcagctactggctagctaatgcttgcaatagtaagttcagtaggcctcattggcccaggcctatctcacatgagggagggctcctttctaagggatgtgacctgatttgtctctgtcatattaccctagagaaggcaagaggagctacagcttaaggaagcacgtgacagaagcgctgaggaagcaaaaacaacagaaatgctgaaagaagaaggctatcagattcagcagaagtgtgaaaagtgtcaccaggtaggaaggaagaatggaaaattcaactcagcctctatcagccagggGTTTCAGGAAATCCTCAAGATGCCCAGCACAGGGTAAAACACACTAAAGCTAGCCTGAGCCTGCTTGTCCCTCATAGCCACATATCAAGCATGTCCTCTATGATGCAATACAGATGTCAGAATCCAACACCAAACAGTTGAAAGCAAGTCCGCCTTCTTCAGATTAGCAAGCAGAACTTTAGTATCCCGAGCTTATCCCCGTCCCGCGGGAAATAAACACAGGACACACCAGATTCTTCCACGGTTAACTT
This mutation is a 970 bp deletion beginning at Chromosome 14 position 41,061,848 bp and ending after 41,062,817 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48078 | GGG TGA GTG CGT GTG TAT TTC | Common | A | |||
| 48079 | TTG TAA GAA AGG GTG CAT GGT | Wild type Reverse | A | |||
| 48080 | TGG CTA TGA GGG ACA AGC AG | Mutant Reverse | A | |||
| 48081 | Fluorophore-1 | AAG AAT GTA TGT CCC TCC CTT CTT | Quencher-1 | WT Probe | ||
| 48082 | Fluorophore-2 | CCA GCA CAG GGT AAA ACA CAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48078 | 0.40 uM |
| 48079 | 0.40 uM |
| 48080 | 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.