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
>chr11:77691431+77691566 136bp ACAACAAACCCTACAATACAGCT GCATTACCGTTTATTTCACCATAG
Mut= 136 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCTACTGGCCCAGAAGATGGACCTGATATGAGTTGTGTCCTAGGTCTGCCTCTGCCTAGACATTGAACCCTGTATCATAATCTGTAAAAGTTTCTTTACATAAAGTTTCTTTACATAAAGATAAGATACAACAAACCCTACAATACAGCTTAgtattgggttcagtaaagttgtttggtcttagtactactgtgatttaaatgtatgtgttttgtttgtttttgttttccttctataggctatggtgaaataaacggtaatgctggagaaagagaaatatctttaaagagcctcagttctgatgaagctaccaaccctatttccagggtccttaatggcaaccagcaagttgtagaaacaagcctgaagcagactgtaaagaccagcacctttggaaaagcaggaatcaaaaccaagaattttattcagaaaaacagtatggacaaaaagaatgggaagtcttatgaaaacaaatctggagagacccaggctgtagataagaccgataccatagcaattccaaatggtgttataacaagtagttcaggctatattactaatggttatatgagcaaaggagcagataatgatggtagtggatctgagagtggatataccactcctaaaaaaaggaaagctaggcgcaatagtgccaagggttgtgaaaaccttaatttagtgcaggacaaaataatgcaagagactagtgtcccagcattaaaacagggacttgaaactttaaagcctgactatagtgaacaaaagggaatgcgagtagatggttcaaagcctatttggaaatatgaaactggacctggaggaacaagtcgtggaaaacctgctatgggtgacgtgcttcgtaaaagctctgatattaaacctggtttgagtagcaaaaaatttgatgatcggcctaaaggaaagcatgcctcagctgctgcctccaaagaggactcatggaccttgtttaagccacccccagtttttccagtggacaatagcagtgcaaaaatagttcctaaaataagttatgcaagcaaagttaaggaaaacctcaacaaaactgtacagaactcctccgtgtcaccatcttcatcttcatcatcatcatctactggggaaactcagacccagtcttcaagtcggttatcccaggtcccgatgtcagctctgaaatctgttacttctgccagtttttctaatgggcctgttttagcaggaactgatggaagtgtgtacccatctgggggtcagccactgctaacgactgctgctaatactttaacacccatctctactgggactgattctgttcttcaggacatgagtctggcttcagcagccgttgaacaaattaaatctagcctttttatctacccttcaaatatgcaaacagtgctgttaagtgcacaagtagatctgccctctcagacagatcagcaaaacctgggggatatcttccagaatcaatggggtttatcatttataaatgagcccagtgctggcccagagactgttattggaaagtcatcagatcataaagtgatggaagtgacatttcaaggagaatatcctgccactttggtttcacagggtgctgaaataattccctcaggaactgagcatcctgtgtttcccaaggcttatgagctggaaaaacggactagtcctcaagttctgggtcacattctaaaacctgggactactgagagtggagccttatccttggaccccagtcatataggtgacctgcaaaaagcagacaccagtagtcaaggtgctttagtgtttctctcaaaggactacgaaatagaaaatcaaaatcccctggcctctcctacgaacactttgttaggctccgccaaagaacagagataccagagaggcctagaaaggaatgatagctggggttcttttgacctgagggctgctattgtatatcacactaaaggtaactcatttgttttttttctaagattccaatgggctgattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtacatgtatatttgatattctatattatgaatgactgggtaagaattagtatcaattaattgttaattgatttgaaatttttgaaaatttattaaagtttagaggactgtagttttccaacgtaaaatcatcaataaaggcacagttgatcacataattagatagaggtttcatgtcccttaaaggatagcctttttttgtaTTGGGTCTCAGGTTTTTTTTTTTTTTTTTCTTTCTTTTTTTTAAGTGATAGAGTCAAAGTTTTAAATTACCCAAATGGGCTTTAAATCCTGATCCTCTACATACTAGTTGCTGCTGGGATTGCCTGTGTATGCCACCAAA
This mutation is a 2080 bp deletion beginning at Chromosome 11 position 77,691,456 bp and ending after 77,693,535 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44010 | ACA ACA AAC CCT ACA ATA CAG CT | Common | A | |||
| 44011 | GCA TTA CCG TTT ATT TCA CCA TAG | Wild type Reverse | A | |||
| 44012 | GCA ACT AGT ATG TAG AGG ATC AGG A | Mutant Reverse | A | |||
| 44013 | Fluorophore-1 | ATT GGG TTC AGT AAA GTT GTT TGG | Quencher-1 | WT Probe | ||
| 44014 | Fluorophore-2 | AAG TTT TAA ATT ACC CAA ATG GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44010 | 0.40 uM |
| 44011 | 0.40 uM |
| 44012 | 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.