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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
>chr9:18380791-18380927 137bp CAAGAAAATCCAAACACAGTGG TCACCTCAACTCCATGCTCA
Mutant= 140bp
Wild Type = 137bp
Sequence
WT sequence; deletion in UPPERCASE
attacagctggttgtgagccaccatgtggttgctgggagttgaactcaggatgtctggaaaagcaatcagtactcttaagtgctaagccatctatccagccccctaaacgtgcctttaatattaaaaaaaaaaggcatgtccaaaaattaatatatccagGTAAGATTTTTTCTATGTCTGACATGTAATTGATACTTGACTGACTGCTCTCTTAATTCTTACTTTTATTCCTCTCATTTTAGTTCCTTTACAAAACTTCCCCATCTGGCAGGAACGGAACAAAATTTACTTCTTGCCAAGAAAATCCAAACACAGTGGAAGAAATTTGGATTAGATTCAGCTAACCTGGTTCATTACGATGTTCTCTTGTCTTATCCAAATGAGACCAATGCAAACTATGTATCCATTGTGGATGAGCATGGAGTTGAGGTGATGTAGCATTATTGAGAGTTTTCATATGTCATAATGTCTGGTTTGCACTTTGCCTTTGTGACAGGATCTCACCTTTTAGCTCTTGCTGGGCAGAAATCTGCTGCATGAACCAGACTGCCTCCAAATTAGTGGCAATCCTCTTGTCTCTGCTTCTTGAGTTCTGGGATTACAGGTGCACACAGTCATGCATAGTTCATTGCTTGCTTCTTGGCTTCTTTATTGATTGGAGTGCCGAGTCAAGtagccttagaattgtgttattagtgcagtaccacttatgtaattcagaaaagttgatacactgccaactcctcagtgtctattcctgttaagtgtggtaaactttctcaggccacgatgaagatttagatatagctctttttctttaagtaaggtgaggcatacaaagggatacatcatttaaa
This mutation is a 514 bp deletion beginning at Chromosome 9 position 18,380,551 bp and ending after 18,381,064 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 67705 | CAA GAA AAT CCA AAC ACA GTG G | Wild type Forward | A | |||
| 67706 | TCA CCT CAA CTC CAT GCT CA | Wild type Reverse | A | |||
| 67707 | CAT CTA TCC AGC CCC CTA AAC | Mutant Forward | A | |||
| 67708 | GTT GGC AGT GTA TCA ACT TTT CTG | Mutant Reverse | A | |||
| 67709 | Fluorophore-1 | CAA TGC AAA CTA TGT ATC CAT T | Quencher-1 | WT Probe | ||
| 67710 | Fluorophore-2 | AGC CTT AGA ATT GTG TTA TTA GTG CAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 67705 | 0.40 uM |
| 67706 | 0.40 uM |
| 67707 | 0.40 uM |
| 67708 | 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.