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
>chr5:138151258-138151353 96bp GTGACCATGGGTCCGAAAG CCTTGTCAGGAAAGGAAGG
Mut= 96 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTGGAGTTCAGTGGTAGAACACTTGCTTGCATGCACAAAGCTGTGGGTTTGATCCCTAGACCCACCAAGAATTTAAAAAAAGAAAACAATATGGCACTCATGAAAGGAAAGACTGGGTGGTTTGGTGGATTTTTGAGACATGTGACCATGGGTCCGAAAGCAGGTAGATTGATTCctagccctggtattgtccttcagctctttcttcttcaaaagtcccttcctttcctgacaaggacagtctgggggacaagatgttggctgctgcgcttctaaaggccaagtctcaggtaagtcatggttcccttcattcttttccttttccttttgatgtctcattcagtgccctaaattctaattcTGTCCATCTAGACTACAGATCTGGGGGCACTGAGATGATGTAGTGCATAAAGGTGTCTGCTGCCATCCTGCGAATCGGAGTTCAATCCCTAGTAGCCACATGTTAGAGAAAACTGGCTCTCAGATTGGCCTGTAACCTCCATGTGTGTACAGTAATAGCCCTTCCCCAAATAAATACATGTTAAT
This mutation is an 186 bp deletion beginning at Chromosome 5 position 138,151,134 bp and ending after 138,151,319 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44724 | GTG ACC ATG GGT CCG AAA G | Common | A | |||
| 44725 | CCT TGT CAG GAA AGG AAG G | Wild type Reverse | A | |||
| 44726 | CAG CAG ACA CCT TTA TGC ACT AC | Mutant Reverse | A | |||
| 44727 | Fluorophore-1 | AGC CCT GGT ATT GTC CTT CAG | Quencher-1 | WT Probe | ||
| 44728 | Fluorophore-2 | AGA CTA CAG ATC TGG GGG CAC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44724 | 0.40 uM |
| 44725 | 0.40 uM |
| 44726 | 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.