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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
>chr2:74859275+74859410 136bp CTTGCTTATCATTTTTAAGGGGAGT AAATGCCTGTGAAGAAAAAGAG
Mut= 124 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATTAAACCATCTTTAAAAATTTGATATTATTCATTTAAGAAAGTAAAGCAATATGTAATATTGCTTACATACACAAATAGAAAATGTAAAATTTTCCTTAAATTTTAAAATTTTATATATTTACTAATGACTTAAGTCTTTTTTTAAGTAAAATATTTTTGTTTGGAAACTTTAAACTTGCTTATCATTTTTAAGGGGAGTACTACTGTATGAACTTTTAAGTTTAGgaaaagaatgaaggaagagttctctttaaatatgcacaagtttttaataaccttattttatttctctttttcttcacaggcatttttgcagatgtgtaatctgcctgtcaaagtggtgtgtagggcaaatgcggaatatatgtctccatctggtaagtgggtgtgttttctattaatacttttgtttgttatttgacattgaggattgaacctaagaccttagctgtgatagcaggtgcattaccactggccttatttctagcacccaacgttaaagtccttagtttgtttgtttgcttgattttttttctgctttgaaggattatttttctattgtttttttaaagtagcagttatgaacttaatcataaagacctatgtgtacttacttgagctctggtccgagGTGTTTTACTGGATTGCTAAGAACATAAGTGATGTCAGAATTCCTTAGTTTTATTTATAGGGCTTTCCTTACCAGGAAACAAATTTTAATGCTGATGAATCTGGTTTTTGTATGGTTTTGTTTGTTTGTTTTTTTATTTACATTCCAGCCATTGCACCCCTCCCCCCTTCCCTGTCGCCCCTCCCACAGTTCCCCATCCCATTTCTCCTCCACTTGTCTCTGAGAGGGTGCTCCGCTCCCACCAAACCAGCTTCCCTGAGGCTTTGTAGTACAGCTTGAGGTCTGGGATGGTGATTCCCTGG
This mutation is a 406 bp deletion beginning at Chromosome 2 position 74,859,326 bp and ending after 74,859,731 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48230 | CTT GCT TAT CAT TTT TAA GGG GAG T | Common | A | |||
| 48231 | AAA TGC CTG TGA AGA AAA AGA G | Wild type Reverse | A | |||
| 48232 | GGT AAG GAA AGC CCT ATA AAT AAA AC | Mutant Reverse | A | |||
| 48233 | Fluorophore-1 | AGG AAG AGT TCT CTT TAA ATA TGC ACA A | Quencher-1 | WT Probe | ||
| 48234 | Fluorophore-2 | AAG TTT AGG TGT TTT ACT GGA TTG CTA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48230 | 0.40 uM |
| 48231 | 0.40 uM |
| 48232 | 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.