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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
Mut= 96 bp
Wt= 113 bp
Sequence
Wt Sequence (deletions in lower case):
CTCATCCTGGCCTCATTTCTGGTGTACTTGGCAGaaaagggTGAGAATGACCACTTTGACACCTACGCAGATGCACTCTGGTGGGGTCTGGTAAGTCCTGGTCACTGGTCACC
7bp del
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39572 | CTC ATC CTG GCC TCA TTT CT | Forward | A | |||
| 39573 | GGT GAC CAG TGA CCA GGA CT | Reverse | A | |||
| 39574 | Fluorophore-1 | TGG CAG AAA AGG GTG AGA AT | Quencher-1 | WT Probe | ||
| 39576 | Fluorophore-2 | CTT GGC AGT GAG AAT GAC CA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39572 | 0.40 uM |
| 39573 | 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.