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
>chr1:80198567-80198682 116bp GATACGGAAACATTTGGCCAT GGGATTGTGGAATAAGGTCA
Mut= 107 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCCAGGACAGCCAGGGCTATACAGAGAAACCCTGTCTCGAAAAAACTTTAAACAAACAAACAAACAAAACTTAGTATGTCCCAGAATGAGAGTGAGCTGTTTGGAGCCAGGACTGATCTTTATAATCAGACTGTTGTCTCATCCCAGCTCAAAATGGCTCCACAGAAAGTCGGCTGTAGTTTGGCACagcagagggtggacaagacacatggtgcttctggaggctaggcagtgaaaaatcgtatttcattggttcttcctttcctgtctcataggtccatccgaagccaggagttggcattaaaaatggtgagcatcccttcttgaacggctgcttgagaggtgacacacatccacaggactccagtctgaactccgtctccacacagagaaccttggagccaaggagccggaggaggagcaggagtcggaggttcaaagtacattcgctggagcttccacaacccagtggctcatgggaaaactctactgaccctttgtggagaaggcttggctggtccaccctggccgacagctccgcgtcaggcatgcagcagcgccgactgtctatccccatagaacccaagatgggaaggaacgttcttagagaagatggctttgagaagctcgaggcggagacaaatgttgacactggcttcaccatcatcaattccgaacccaggcgatcttttccgagcagatttcccagggattttcagagcagccaaccaccacgctgcttgtcaggctcttccttagaggtaactgtttccccaaaccaaggagtctttaaggatagactccaccctctgtcacttcctagtcaaagggactttggtgcaaagaaggtcatctcaaaatgttcccatcatctgcaggcccagggtgaagagaaagaagaattctttatatagcataaaacaattgtggttttttttttcctaagacacgagaatgttatagaaaccaagcgttgtattagttgcctttgtagttgaaatgagaaatatcctacaaaagcaagcaaaggtttaagagtcagccccattcatggctgggaagtcacagaagcaagagtgtgactggttacgtttcctctacagtcaggtagcagagggggatggatgctggcgatctgcctgcctttttaaaagctcaatctgggactccagctcatgggaagatattacccatttttagggttggtcttccttcctcagttaaatctttctggaaacacccttctagatacacccagagatgggtttccatggtgtgtctgaatccagttgacagtgaagatcggatctacgaacatttagtttcttgctcctttgagagagatctaggctctcgatgcccagtattagaagtccacggggatctgacagtgtgctggccagttggaagggagagaaagaaaacccagggtctgagcttctcagagacacagctcagatgcatgtgttggctcagcacctagcagggccttggcactgtgtggaaatgaatcagatgtgctatgcaaatctgattagtgtgtgtaatatggttttaatttttccttgaggttgcatagcttatccccttggacagtaggaactatttattctttagtgcgttttaatttttgtgtggcctcctttggtaagtaggccctgtttacgatacaccctcttgtggttttgcctttaaagttctttgcatatgaaacgagtgaagaatgcaaagtgttttaaaattataaataccaccccctcccctgtttaattagtaacaatactcgaagccagaaagctaggttcaaaatgagtgctagactcagagatatttctactcttttgtctctcatttgttcatgtcctacttgaaagagatcatcaggtcttgttaaaacaaaaacaaacaaacaaacaaacaaacacaaaaaaacaaacaaaacaaaaacaaacaaacaaacaaaaccaaggaagacagggatgattagaaaattctgtatctccactgagcatgccgaaaggttgtgttggctgagttgctagaccaggacttgagttctagctgtattttgggggctcctggggcttgtatgttcacgtaataaacatttttttttgcatgcagtaaatattcatggatatgatacggaaacatttggccatcaaattcaaaaagtattcatggctttgcaactgcaaccctccacTGAACCAACTCTTGAAAAAGGTGTTTAAGTTTGACCTTATTCCACAATCCCAGCCCACATTTGCCATGTAAAGATGTACTAAACTCTTAGTTTTCTCATCGTTTTCATGAAACACCCCAGCAAAAGCAAATTACGGTAGGGAGGGATTATTTTGGCTGGTAGTTCCAGGTTAGAGTCTAT
This mutation is a 2019 bp deletion beginning at Chromosome 1 position 80,198,618 bp and ending after 80,200,636 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44540 | GAT ACG GAA ACA TTT GGC CAT | Wild type Forward | A | |||
| 44541 | GGG ATT GTG GAA TAA GGT CA | Common | A | |||
| 44542 | TGT TGT CTC ATC CCA GCT CA | Mutant Forward | A | |||
| 44543 | Fluorophore-1 | AAA AAG TAT TCA TGG CTT TGC AAC | Quencher-1 | WT Probe | ||
| 44544 | Fluorophore-2 | CGG CTG TAG TTT GGC ACT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44540 | 0.40 uM |
| 44541 | 0.40 uM |
| 44542 | 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.