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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
Mutant= 86 bp
Wild Type = 129 bp
>chr2:106967341-106967469 129bp CAACCATTGCTGTCCTCA TACACACAGAAAGGAAGGTG
Sequence
Wt Sequence (deletion in lower case):
ATATATATGTATATACATGTGTATGTATAGACACAGACACAGTACACCAGAACGTGAAGCCACTGTACTTCAAGTTAGAAGGTCTGAAGGACACCTCAGTTCTGTGCTGTTGGATTATGTGACACTTTCTGCTGCCTTCTCTCCTGAGGTGGCAACCATTGCTGTCCTCAGTGTGCCAGGCTCCTGCACctgctgtctaagagactaaagtagcaaaaataatacaaaagtttgaatatagactagaagagtggcttttcccaccttcctttctgtgtgtattccaaactgcattccagactcggggggaggggggtgaccctgggctagtgctaagtaaagtcctattgtttgaccccaagattttaatttatagttttcttactttattcagggaaggactgctaaagctctgaggtcactacagtttacaaacccagggaagcaaactgaatttgctccagaaggcggtaagagggagaaaagaaagctcaccaaagccacaagcgctgcttctgacaggtgggcagccagctccgttctctccgctatttgttacattcctgctgcacgtccacattatccttgtgggttttttcttaattttcagttttctgaccaatcattttaaattgtactctgattagttaaacctgaaagccatGGCTGTTACTCCTAGGGAAGTAAAGCGGCCGTTGGAGGTCTAGAAACTTCTTTCTGAGAAGGTAGCATGAAGTGAATGAGGTAATGAGAATAAGAGTTAAAATAATTGAAGAAACATCTTAGAACTCACCAGTCTCTAAGATGTTTACTATTTGCAAAATTTGATATTGATGTTCTCTTCTTGATGTGTCTGCTTTCTGTGTGCAGCTGACAGAAAGGCCATTGCATTTATGACAACTCAAGAGTG
This mutation is a 475 bp deletion beginning at Chromosome 2 position 106,966,958 bp and ending after 106,967,432 bp (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68965 | CAA CCA TTG CTG TCC TCA | Common | A | |||
| 68966 | TAC ACA CAG AAA GGA AGG TG | Wild type Reverse | A | |||
| 68967 | AAG TTT CTA GAC CTC CAA CG | Mutant Reverse | A | |||
| 68968 | Fluorophore-1 | GCA CCT GCT GTC TAA GAG A | Quencher-1 | WT Probe | ||
| 68969 | Fluorophore-2 | CTC CTG CAC GGC TGT TAC TC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68965 | 0.40 uM |
| 68966 | 0.40 uM |
| 68967 | 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.