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
Sequence
Wt Sequence (deletion in lower case):
TAATTGTTACTTTTAAGTTTTGAAATTCATTCTTAGCTTATATGTGAAAAGAAGCCAATGAAATCATGCTGATCCTTGGATGTCTTTCTCTTGCTTGCCCCCTCCTCCCCTTCCCCCCCTCTTTCTCTCTCTCTCTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTGTAATAGCGAgttgcgccagccttgatagacttcttttggagtttaatatggtgaatatttttatctcaaagtgtataactattcatgaattgtcatgcttgaataatgatttaacagttatatttgtttcccttggacttataaaaatttattataacatttatattataaaatttaatgttaatgtttttctccaattacaggagtcagaaataccatcagaggaggggtactgtgactttaatagcaggccaaatgagaactcttattgctatcaacttcttcgacaactagatgaacagagaaagaaagatattctttgtgatgtcagcattgtggtgagcgggaaaatctttaaagctcataagaacatcctggttgcaggcagccgtttctttaagactttatattgtgtttcaaacaaagaaagccctaaccaaaacaatactacccacttagatattgctgcagttcaaggtttttcagtcatcttggacttcctgtattctgggaacctggtgctcacaagccaaaacgccattgaagtgatgacagtggctagctaccttcaaatgagtgaagtcgttcaaacttgcaggaatttcattaaagatgccttaaatatcagcattaaatcggaagctccggagtctgtggttgtggactataacaataggaagccagttagcagggacagcctgccttcatcctcacgggaacagaagatcgccagcttctgggcaacgcggaacctgaccaacttggcaagtaatataaaaactgaaaacgaaggctgtgatgtcgacgagggccaagtagaaaactaccagatgaatgacagtaactggatccgggatggctctcctgaattggctgaaaatgaatctcaaggtaaaacaaaagtgtttatttggaataatatgacctctcaagagactggcaaagcaaggaggaaaaaccaaactacaaagagatttgtttataatataccgcctaacagtgagacaaaggtagaagattgctcagtgatgcagccccctgctgcctacccagaagaaagcaaggccttactcatcaaggaagagccaggtaaggttgtcagtccagttttgtacctgctgaagtttacttttaattattaatgtattagaactaaggatacttagttcttagttttgggtcttttgtgtgtctatgtgagtttaagttttagagtgggtggtaactaaagtaccacctgccaaaagccttattaataactatgtaacagactctttctgacttagggtaagGTTGTATCTCTGTAAACACCATAAGATCACTCACGCTGTCTTTAATCTTTAAGTCAGAATTGTTAATATCCTCAGCCCATTGTATATCACAGTTTGAAGAGCTTTCTTATAATAAGTGATAACTGTGTTATGTAAATTTATTAGATGCTAACCGAAACTAAAAGTGATTTTAGGGTTTGCCTTTTCATAATTTGGAAACA
This mutation is a 1,283 bp deletion of Chr3:9,264,341-9,265,623 (GRCm38/mm10)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 69784 | AGT CTG TGG TTG TGG ACT AT | Wild type Forward | A | |||
| 69788 | Fluorophore-1 | CAA TAG GAA GCC AGT TAG CA | Quencher-1 | WT Probe | ||
| 70452 | GAT CTT CTG TTC CCG TGA G | Wild type Reverse | A | |||
| 70453 | TCT TTC TGT AAT AGC GAG TTG T | Mutant Forward | A | |||
| 70454 | TGG GCT GAG GAT ATT AAC AA | Mutant Reverse | A | |||
| 70455 | Fluorophore-2 | CAC CAT AAG ATC ACT CAC GCT GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 69784 | 0.40 uM |
| 70452 | 0.40 uM |
| 70453 | 0.40 uM |
| 70454 | 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.