For in-depth product & services help, ask our
Technical Information Scientists
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= 78 bp
Wild Type = 95 bp
Sequence
Large deletion: Mutant sequence with junction in uppercase
atcatctgctctctgcgtctcggacgctatttaaagcGAcaagggttttggtagccagcactccacacctgcaagaga
This mutation is a 3243 bp deletion beginning at Chromosome 15 position 98,167,142 bp and ending after 98,170,384 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62892 | ATC ATC TGC TCT CTG CGT CTC | Common | A | |||
| 62893 | GAA AAC TCC ATA ACA ACA GAT TCC | Wild type Reverse | A | |||
| 62894 | TCT CTT GCA GGT GTG GAG TG | Mutant Reverse | A | |||
| 62895 | Fluorophore-1 | ACG CTA TTT AAA GCG CTG GG | Quencher-1 | WT Probe | ||
| 62896 | Fluorophore-2 | ACG CTA TTT AAA GCG ACA AGG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62892 | 0.40 uM |
| 62893 | 0.40 uM |
| 62894 | 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.