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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
>chr9:44102579+44102694 116bp ACTACCACCCCAGCAAGGAC CTTTCCTCCCCAACACCATC
Mutant= 112 bp
Wild Type = 116 bp
Sequence
Wt Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTaagtCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG
Mutant Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38489 | ACT ACC ACC CCA GCA AGG AC | Forward | A | |||
| 38490 | CTT TCC TCC CCA ACA CCA TC | Reverse | A | |||
| 38491 | Fluorophore-1 | CAG ACC AGT AAG TCC CAA GGG | Quencher-1 | WT Probe | ||
| 38492 | Fluorophore-2 | CAG ACC AGT CCC AAG GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38489 | 0.40 uM |
| 38490 | 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.