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
>chr10:13559852+13559973 122bp CCCAAGGTACACAAGAATTTCA GTTTCAGGATGCCCTTAGCA
Mut= 114 bp
Wt= 122 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TTCTGGTAAACTCAATAAGGCTTTGCTTTAGAGTGAGGTCAGTTGAAGATATATTTGAAACTAGAGCATAAGCTTATGAAATTTGAAATCTTCCATTAAAATATTCAGAAATGGTAGGTCTTTTTTTTGAGACTTATCTCCTCTGTAATAATTTAATGAAAAACAAATTTTCCCAAGGTACACAAGAATTTCACTAAAAATATAGCAAGGTTTGTGTAGTACAGTGGATTAGACATCCCTgttggtgagtttatgatatttgcactggggaactgctaagggcatcctgaaacccatgttctaatacagtcagaaatggtaggaatgccttttgaattgacattttgcatattttaaagacaccagctgtacgaaacaaacaatgtcaatacaactctctctctctcccccccccctctctctctctctcaaggctactcggcatgctgaaatggtggccattgatcaggtcctagactggtgtcatcagcatggacagagcccttccactgtattcgagcacaccgtgttgtacgtcactgtggagccgtgcatcatgtgtgctgccgccctgcgccttatgagtatcctttggcttctagacttcctgcctttaccttgcctgtgagcacaacatggccagattcacgacccattgttctttctttttagtgcttgtttttcttttcaacgttgagaatggaagccaggctcttGTACTTGCTGCATCTGCTCTGTCCCTGAGCCATAGCCGTCCTTAGCTTTCAGTTCCTTAATGTGTGGAAACAGCGTTATGAAATACACTCTACAATTTAGCCAGAAACAGGAAAACCAGTTTCTACACAGCAGAGTGACCTGTTCTTTCTCTGTATCGACAGCCATGAAAGAATTGAATACTTAAGCAAAGGGAAGAAAAGCTCTTTTGTCAGGATAAATCCAAATTAAGGTGGAGCACAGG
This mutation is a 476 bp deletion beginning at Chromosome 10 position 13,435,665 bp and ending after 13,436,140 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64690 | CCC AAG GTA CAC AAG AAT TTC A | Common | A | |||
| 64691 | GTT TCA GGA TGC CCT TAG CA | Wild type Reverse | A | |||
| 64692 | CTA AGG ACG GCT ATG GCT CA | Mutant Reverse | A | |||
| 64693 | Fluorophore-1 | TGG TGA GTT TAT GAT ATT TGC ACT G | Quencher-1 | WT Probe | ||
| 64694 | Fluorophore-2 | AGA CAT CCC TGT ACT TGC TGC ATC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64690 | 0.40 uM |
| 64691 | 0.40 uM |
| 64692 | 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.