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
>chrX:134266357-134266456 100bp GTGAGGGGGAATGAGAGTCA GCGTACTGGGCACCTACTATTC
Mut= 97 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ATAGTCCCTTTACAGCCAAACACAATATGTCTCATTGTATGTGACAAATGTATTTTTAAGGTCAAATTATATTTTAAAAGCATTAAGGAGGTGAGCTTTTGAGAGGAACTCAGAAGCAAAGCTTTTTGTGATATGACTAATTACCGCCTGATAAACCTGGACTTTTCTTATATTGAATTTGCCCATCTGTTCTGCTTTGTGAAATACTAGATAGACATCACCCGAATGGGCTCTTCTCCCAGACTCGTGAGGGGGAATGAGAGTCAGTTGTATCTTCTGAATTTAAGTAAAACCtcagagagtttgaggaccggggctatttacgaatagtaggtgcccagtacgcatttagataattcacagtgtatctgaattattttgttcaaggctggctgatgttgtgactccactctggaggctgagttatgaagcacaactcaaggtaggtgtctagcttgagtcttctcccccagctcctccagcccaatggcacTAGAGTCACGGAGAGCCTGATTGGAGACTTCAGTCCACTTGCCACATAGTTTCTTTCTTTGTTGTTGTTGTTTGTTTGTTTTTGAAGCCAGGGTTCCTGTAGCCCAGGCTGGTTCCAAGTCCCTAAGTAACTAAGGATGACCTTGAACATGTTGATCTTCCTGCCTCCTGAATTATAGTTGTAGACTATCACACTTACTTTATGCAGTCTCTGGCCTTAAACCCAGGGTTTCCTGCAGGCCCCACAAGCATTGTGACAAGGAAATCCTGTTCCCAG
This mutation is a 199 bp deletion beginning at Chromosome X position 134,266,210 bp and ending after 134,266,408 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48084 | GTG AGG GGG AAT GAG AGT CA | Common | A | |||
| 48085 | GCG TAC TGG GCA CCT ACT ATT C | Wild type Reverse | A | |||
| 48086 | CTA TGT GGC AAG TGG ACT GAA G | Mutant Reverse | A | |||
| 48087 | Fluorophore-1 | AGT TTG AGG ACC GGG GC | Quencher-1 | WT Probe | ||
| 48088 | Fluorophore-2 | AGT AAA ACC TAG AGT CAC GGA GAG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48084 | 0.40 uM |
| 48085 | 0.40 uM |
| 48086 | 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.