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
>chr6:58907072-58907168 97bp AGTGAGCAGCCATTCACTTG GGTCCAGAGTGCGTCTGAAG
Mutant= 94 bp
Wild Type = 97 bp
Sequence
GGGGGGGGGTGTATAAACAGGTATGGAAAAATACAGTCTCAGCCTCGAGCTGGTTCCAGCCAGTTCTGCAGGGGTGGGGGCGGATGCTGAGGGCTGGGTGTCTAAGTGGCAGGTTTTAGAGACCTCGGAGATAGCAGCATAGCAACAATTTGGTGCAGTGTGTGCGCACAACCATCAGCTGATAGTGAGCAGCCATTCACTTGCCGCGAGCcgcgaagcggccgcgcggacgtcaccacagccgtcagcgtttgccgcatcttcagacgcactctggaccagccgcaatcacagctgcaggacctctctggaccagctctattgcagactgcgcaaccaccagaccactgcggcaaacaagcccagctgagccgagcagtagccatggccgaccccgagaagcagggacccgctgagagccgcgccgaggacgaggtaatggaaggcgcccagggtggcgaagatgccgcgaccggtgacagtgccgctgctcccgcggccgaggagccccaggcccccgcggagaacgcgcccaagcccaaaaaagactttatggagagcttgcccaattccgtgaaatgccgggttctggcgctcaaaaagctgcagaagcgctgcgataagatcgaggccaagtttgacaaggaattccaggctctggagaagaagtacaacgatatctacaagcccctgctcgccaagatccaggagctcaccggagagatggagggctgcgcgtggaccctggagggagaggatgatgaagatgacgaggaagaggacgacgaggaggaggaggaggaagaagaggctgcagctggcgcaactgggggtcccaacttcgccaagaagtgagcacagcaagctgacaggtgataaatcccctgccccttttatttccaatgtcggcagactttaaaaggctcaggatttgcccaaaataaaatgtgaatctattctgacattcctaaaattaaattgaatcaagtagattctggctagcccgtttctaatctgacttttatttgtgtaaaacagatgctaaaagacgttttttagaaaaaaaaaaaagcctaattaaacttttttttttctccctgctcttctgtgagaactggacttgagactgttgaaaggttgtcagttagatgggaattaaaagtgcgtcacacgttgaaatccattcatcacactacaccagaacatccaagctaagacagaactcttctcaatgcttaattcttcagtttctttacattttccagcgcagaggaaaaggaacccaggaacgacgtcatctttaagacttttgcttttgcaaacccagacatcagctttacactccagaggagccttggcatggagcaagcctggactgacagcatttactgattatgtggctaggaaaactgccatttcaagttgtgaaaaaatgttttaaatatttgcatttacagaaagaacatggtttcaaaaatgagggtgtattccatgtataatattgtcaacacgtgttcatctgttatggtcccgtgttacctgttttcttggtagtgtttgtttacaaaatcgtaaaaaattaccccaaatgttttaaagtgttaaattaccttatagcattttagaaatataatttacttgaagagatgtagaatgtagcaattctgtaaagcatgtgtatccagtgttgcctagtttgacctttgtgaagtctttttgtcttgtagcttttagcaagtagctgtgaaaaccatcagaactcctcaatgaagctaatgtttggaaaaagtatatacttgaagaacaaacccaagtgtgtacccccaaccccagctcagaaatagaaagggtttaagtttgcttgtattagctgtgccttcattattttgctatgtaaatgtgacttaataaatggtgcaaaattaaattttattgcttgaggacagacgggcataaaggaagggaaaagacatttaatgtaaaggcctctagcttctttgtgggttttgagttgtctgtgaaccaatgttctgtaaagaaacacaaacttaaagttgtttaccactgtggtgttaacaaaacagtattttcaaaaataaaaaaatccttgttattctgactacctgtactgtcatctataaaagtgggtgggaacaatcccttcctcgcagttattatgaagagaaagtgagtaactacgcttacagcccacacagcagcgtctgacacctaggaataatttccagctattattgttttaataaagacaaaagacccagatagaaaattcaactccctcccctgctacacaacagaaaatggtagcattattaatttctaacttggtgattccttgttatcagaaaagaatatggcTTGTAATTTAGAAATATAGGACATTCAAAAACTATCTTGTTTTAGTTTCCTCCATATAAAGCCAAGTTTTAATTTCCTCTTAGACAAGGTAAGATTTTAATGTGACAATATGAATGCACACACACACATGCACAAACAAACCAAAAAATAATCAGACGCCATTCTTTTCCATTTGCTGCAAGGGTTAATCACACTGCAGCAAAATCT
A 2165 bp deletion beginning at Chromosome 6 position 58,904,976 bp and ending after 58,907,140 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44798 | AGT GAG CAG CCA TTC ACT TG | Common | A | |||
| 44799 | GGT CCA GAG TGC GTC TGA AG | Wild type Reverse | A | |||
| 44800 | CTT GGC TTT ATA TGG AGG AAA C | Mutant Reverse | A | |||
| 44801 | Fluorophore-1 | CGC GAG CTT GTA ATT TAG AAA TAT AGG | Quencher-1 | MUT Probe | ||
| 44802 | Fluorophore-2 | TCA CCA CAG CCG TCA GC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44798 | 0.40 uM |
| 44799 | 0.40 uM |
| 44800 | 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.