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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
>chr1:71353716-71353874 159bp AGGTGAGAAATTTGCAAGTCC TAAATTGCTGTCCCTCTTCG
Mut= 131 bp
Wt= 159 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GACTAATTTAAAGTCTCATCTAGTCCATTATCAAAGAATAAAAGTTTACCAGGCAAGAAACCTTATATGCTCATTGGTACATTGGTTAAGTGACCTCATCCTTTAATATTCTAGAAAATAGGTGAGAAATTTGCAAGTCCTTGGAGCCCTGAACCTTCTGCATAATACATTGCTTTAGTCAACTGAAATTAatgtggacaaagtcagagagttcttgctttttcttctctagcgaaattctgaaaaagccgtccaacccgaagagggacagcaatttatcactccggagcacccaagttccagaaagaagtcacacatcattgggtaagccagctctctcttgtaaggcttcgaatagatttttggaacagtcagcaaaagggatactctttaaaaggtttcaatttcacattttcaaataaaaatggagtgcttagattgccccCAACGGACCCCAGGATTGCCCATGGCTGCCTGAATGTGGCAGTATGGTTGTGCAGAATTCTTTCTTCAGAACAAGTAGAAATTAAGTTCTTACTGAAACGCTCTATAGGAAGGAACACCTAGATATTGAGAATTGCATCAAGTCCCAGAGTTGAGTCAGGACTTCACCTGCAAAATAAATAGAACTGTGGTGTATGGAATTTCCATCA
This mutation is a 254 bp deletion beginning at Chromosome 1 position 71,353,549 bp and ending after 71,353,802 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46911 | AGG TGA GAA ATT TGC AAG TCC | Common | A | |||
| 46912 | TAA ATT GCT GTC CCT CTT CG | Wild type Reverse | A | |||
| 46913 | AAT TCT GCA CAA CCA TAC TGC | Mutant Reverse | A | |||
| 46914 | Fluorophore-1 | AGC GAA ATT CTG AAA AAG CCG | Quencher-1 | WT Probe | ||
| 46915 | Fluorophore-2 | CTG AAA TTA CAA CGG ACC CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46911 | 0.40 uM |
| 46912 | 0.40 uM |
| 46913 | 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.