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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
>chr16:13111959+13112099 141bp TTGTCTGTCTCCTCTGCTAAACG CTCAGTGTTAAGTGACATGTAGCC
Mut= 141 bp
Wt= 141 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAGAAAACTTTTAACCCAAAGTTTTAAAAGGTTCTGTGGCTTGGTGTTGAAATGGAAATAATGACTGGAGCACAATTATTTCTTAATATAAACTCCAAAGCACAACTTAACCTTACAAAACAAAACCTTAATTAAAGTGACCTGATTGACTGATGGGTATAAAAGAATAACTTAGTTTGCATGTTTGCTGTGATGTTCATAAGCTCTGAGAATGGAGACATCATTTTAAATCCCTATAGCCTATTTTAAAACTTCCCTGccctgtactaagcagcttactaatgagtttaatttgatttaggaatattttatcaatcagttgaagatagaaggagttgaacatctccctcgacgagtgacaaatgaaatcgcaagtaacagtcgctatgaagtctacacgcagggtggtattatatttgcaacaagccgaatactcgtggttgatttcttgacaggtagaataccttcagatttaataactggtaagaatttgaaatctttcttatctacaatgtgaacatttttatttttgtctgtctcctctgctaaacgttctgcttttgttactcagtattgtagctaagctgttgacacactacctgacaagtcgagagGTTCAGCAAATGTTGATGACATGAATAAGTATGGCTACATGTCACTTAACACTGAGAATCTATGATTTAGCTATTGTATGAAGGCCTTAGCGCTTACCGACAAAATCCCGCATGGGTGGCCTGGAGAGGTGGATCGGAGATTAAGAAGGTGCACTGCTCTTGCAGAATTCTAAAGCTTAGTTCCCAGCACCCATGTAAGGTAGCTCACAACTGCCCATAATTCCAACTCCAGGGCATCCGATTCCCTCTTTGGCCCTCTGAGAGCACTGGACTCCACCTGCACAAC
This mutation is a 355 bp deletion beginning at Chromosome 16 position 13,111,689 bp and ending after 13,112,043 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48151 | TTG TCT GTC TCC TCT GCT AAA CG | Wild type Forward | A | Wt F | ||
| 48152 | CTC AGT GTT AAG TGA CAT GTA GCC | Common | A | Common | ||
| 48153 | GTT TGC ATG TTT GCT GTG ATG | Mutant Forward | A | Mut F | ||
| 48156 | Fluorophore-1 | AGC TGT TGA CAC ACT ACC TGA CAA | Quencher-1 | WT Probe | ||
| 48157 | Fluorophore-2 | AAA ACT TCC CTG GTT CAG CAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48151 | 0.40 uM |
| 48152 | 0.40 uM |
| 48153 | 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.