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
>chr1:127888946+127889051 106bp GGTCTTCAGGCATCTTGCTG TGCATTCGACAGTTATCCTCT
Mut= 106 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACTTATTTGTCTGTGTTTATGTGTGTGTGTTTGTGTGTGTGTGCACAGACACGCATGCGTGCATGTATACATGCTGATGTGTGTGGAGGTTAGAGGACAACTTGGAGGAGTCAGTTCCCTCTTTCTATGTGGTTTCCAGGAGTCAAACTTAGGTCTTCAGGCATCTTGCTGACTCCAAATagccactgtgataagtcatttattctcctgaaattatttttttgcctttatcatgtagaggataactgtcgaatgcagtgctatgattaatgttagaaaagagacaaaaatcattgcatagtaaacactatgatgactgttgagtaagctggctgttgtttctttctcttgttaaatgcagggtatatttacttctggcacatgggaagagcgatcagatgaaatttcctttgctgacttcaggttttcagtcacacatcactatcttgtacaagagtctcctgataaagaaagaaaggatgaagaattggagggtatggggtttttgtttttgttttgtggtttgttttttttttggagggggatcatatttgtacaatctttaaagagtctaatttttagttattagtttagttctcataactcaataatacccataggaagcatttttttttgtaaccacatttgaaatagcttatgtgctagtactgcatctgaagAAGAGGAGAAGCCACAGTGACGAGCACTTTAGACTTGATTGAACCAGCAGATATTGACTGAGTGGCTCATTTGGTGATTGGTGGTAGGTCATGTAGTCTACTCTGAGATGCCCTATGTGTAACAGTGAGCAGGGGGTTTCAGCCGCTAGTCAAACAGTAGTCATTAGTCCTCCTGTTGTC
This mutation is a 500 bp deletion beginning at Chromosome 1 position 127,888,975 bp and ending after 127,889,474 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46807 | GGT CTT CAG GCA TCT TGC TG | Common | A | |||
| 46808 | TGC ATT CGA CAG TTA TCC TCT | Wild type Reverse | A | |||
| 46809 | TCA CCA AAT GAG CCA CTC AG | Mutant Reverse | A | |||
| 46810 | Fluorophore-1 | AGC CAC TGT GAT AAG TCA TTT ATT CTC | Quencher-1 | WT Probe | ||
| 46811 | Fluorophore-2 | CCA CAG TGA CGA GCA CTT TAG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46807 | 0.40 uM |
| 46808 | 0.40 uM |
| 46809 | 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.