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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
>chr12:69958835+69958960 126bp GCAATAGCAGTTGAATAGGCTGT GAATGCTTCCTAAGAGTGATGGA
Mut= 103 bp
Wt= 126 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
ttttaataaagtagaaatgggctctaaaatactgttgagagaagtgtagtaaatataatcatttattatgaaaattctgcactgcatgaattgtatacgttatactttaagcaatagcagttgaataggctgttcatagtaactgtgctctgactgacaacacagttctggctgcagtgtctcgaggtggtggaagtgtagcccaccccttagtccatcactcttaggaagcattctggtgtactccaagaaaaggcatggacacagttgttaatttactccatattacataaacagaagcctctctttctgacagGTTTGTGGTGGTGATAAACCATTTTTGGCCCCTAACGACCTGCAAAGCAAGCACCTGCAGCTAAAAGAAGAATCTGTGAAGTTATTCCGAGGCGTGAAGAAGATGGGCGGGGAAGAGTTCAGCCGGCGTTACCTGCAGC
Mutant Sequence:
AAATACTATGTTACTGTACCGAACTTTTTTAATAAAGTAGAAATGGGCTCTAAAATACTGTTGAGAGAAGTGTAGTAAATATAATCATTTATTATGAAAATTCTGCACTGCATGAATTGTATACGTTATACTTTAAGCAATAGCAGTTGAATAGGCTGTTCATAGTAACTGTGCTCTGACTGACAACACAGTTCTGGCTGCAGTGTCTgcactgGCTTGGTGAGGGGCATATGTGAACCACAATCATTTGGTTATATGTCATTCCCTTTAAGTATCAGATTACTTCAGACTGTAATTGACCTATACTTCTGCACTAAGCTGCAGCATGAGCCAGTGGTCAGCCTCTGACGTAGCTGCCTG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 60689 | GCA ATA GCA GTT GAA TAG GCT GT | Common | A | |||
| 60690 | GAA TGC TTC CTA AGA GTG ATG GA | Wild type Reverse | A | |||
| 60691 | GGT TCA CAT ATG CCC CTC AC | Mutant Reverse | A | |||
| 60692 | Fluorophore-1 | AGG TGG TGG AAG TGT AGC CCA | Quencher-1 | WT Probe | ||
| 60693 | Fluorophore-2 | CTG CAG TGT CTG CAC TGG CTT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 60689 | 0.40 uM |
| 60690 | 0.40 uM |
| 60691 | 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.