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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
>chr19:8873347+8873439 93bp CGTGTGTCACCAGGTCTGGC ATAAGGGAATACGGGCTCCA
Mut= 98 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAAAGGCGTGCACTGCTACCACCTAGCTTGTCCTCTTTGGTTTTTCAAGATAGGGTTTCTTTGTATAGTCCTGGCTGTCCTGGAACTAACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTTCCAAGTGCTGGGATTAAAGGCGTGTGTCACCAGGTCTGGCCCTGCTTgtcctctttatattactgttttggttttgtttcctttcctctgtcttggagcccgtattcccttattttgttaatttctcagtctaaactcaccttcccactccatgtccaactttacctctttcttctctgcaaaacagaatgttggaacttgtggcacaaaagcagcgggaacgtgaagaaagagaggagcgagaagctttagaacgagaaaagcagcggaggagacaagggcaagagctgtcagttgcacgacagaaactgcaggaagatgagatgcgccgggctgcggaggagcgcaggagggaaaaggctgaagagttagctgccaggtctgaagtctcattggtcgctaattgggaaagaaatgaagatttgccttgtccatgtctgacctattttcttcctgtctctattctagacaaagggttcgagaaaaaattgaaagggacaaagcagagagagccaagaaggtgggtgattagaaagtctgggtatagtggagtatgtgggttaatttccttaaagtgtgcttagcatgatttttttgaggcaattttactctagcccagactagtgtcagactcatagtattctcctgcgtcagccttccaagtactctaactaaaagctgtgaactgccaaacctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttggcccatgtactggagggtgaccctttatacatgctagactagtgtgctccctctgaggtaGTGTTCCATTTTGTCTTTCAGTATGGTGGTAGTGTGGGTTCTCGGTCATCCCCACCAGCAACAGACCCAGGTCCTGTTCCTTCTTCTCCCAGCCAGGAGCCCCCTACTAAGCGGGAGTATGACCAGTGTCGTATACAGGTACTGTTTTCAAGTGACCGTCTTGCTTCTGGAATCCCGTGTTGGCTTATAAAGT
This mutation is a 744 bp deletion beginning at Chromosome 19 position 8,873,374 bp and ending after 8,874,117 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47742 | CGT GTG TCA CCA GGT CTG GC | Common | A | Common | ||
| 47743 | ATA AGG GAA TAC GGG CTC CA | Wild type Reverse | A | Wt R | ||
| 47744 | CCT GGG TCT GTT GCT GGT G | Mutant Reverse | A | Mut R | ||
| 47745 | Fluorophore-1 | TGG TTT TGT TTC CTT TCC TCT G | Quencher-1 | WT Probe | ||
| 47746 | Fluorophore-2 | CAG TAT GGT GGT AGT GTG GGT TC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47742 | 0.40 uM |
| 47743 | 0.40 uM |
| 47744 | 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.