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Notes
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 = 103 bp
Heterozygote = 153 bp and 103 bp
Wild type = 153 bp
This assay is capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 14.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
This protocol can be used for conventional PCR. Omit probes and run as a separated assay.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 78122 | GAT GTG AAT AAA AGG CGA CA | Wild type Forward | A | |||
| 78123 | ATG TAA TGA ATC ACC AGC CT | Common | A | |||
| 78124 | CAG ATC AAC ATC CCT GAA GG | Mutant Forward | A | |||
| 78125 | Fluorophore-1 | ACA AAG CCT ACA CAA TAG CC | Quencher-1 | WT Probe | ||
| 78126 | Fluorophore-2 | CAA ACA GTG AGT ATT AAG TGT CTG GT | Quencher-2 | Tg Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 78122 | 0.40 uM |
| 78123 | 0.40 uM |
| 78124 | 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.