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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
Mutant= 90 bp
Wild Type = 109 bp
>chr4:96769872-96769980 109bp ATGCTTAGACAGACAGCCATGT ATACACAACGCTGGCATGAG
Sequence
Sequence (deleted region in lower case)
ACTGGCAAAAGAGTTTGACTTTTGTCCATTTCTAGGTGCAAAAGCTTATTTGAGTTATAAATGCGCAATGTTCTCATGGTTAGGGTTCAATTTTGACTGTGTTCACTTCCTGTAGTAAGTGGTCTAGAGCAGATAATCAGATTATTAGGAAGTGAGAATAGAAAAAATAGTCTGAGAAGACAGTGGCATGGTGCTCTCAGGTACATGATGCTTAGACAGACAGCCATGTTTACCACTAAttacatctcacagggctgttcctgctgttcacgcttccatactcacaccttctgcttctcatgccagcgttgtgtatgatgaggagtgttccttatgactgacaaggtgcaaactctcttcacagtaagcagaatgagaagttgaagacagaacctcaaacctcactccagaaacccaggaatgattattcaagacgggtgagcagagacctgccaggacccactgactcttctgagcaacaaataactgctaacccagctgagaaggaagaaagcaagcaccaaagaagcagcctaaaacccgaaagcaatcaaaagttcttaaccagagtattcagtaacaggttcctggatggcagggtgagtaacaagagatggccaagcttttcttggtaactcattagaaaagtgcagaggagaacataatttcatgcatacatatacaaatagaaataagtactttttcatagactagaaattataaatgcttctttttcaacttgaaaataaattttgaggggattttgtgtagtacaattttgtctacaacaaattgagctggtcaacataactgtggcaaACTCACTGATACTCAGCAATGGATACAGTAATGAGCATCACTAGCCAAAGGTCTGAGGTTTATGTTATGTTACGTGATAAACGCCATGATCAAAAGCCACTTAGGGAGGAAGAAGGGGCTTATTTTTCAGACAGCTTATGGTCCATCTTGAAGGTAAATCATAGTATAGAAAGGCAAGAACCTTGAGTCCTGAACTGGAGCAGTGACCACTGAGGGATGGTGCTTATTGGTTTGCTCTCCGTGGCTTGCCCAGCCTGGTCTCTTATATAACTCAGGACCACCTGACCAAGGTAGCACCATTCCAAATGAGC
580 bp deletion beginning at Chromosome 4 negative strand position 96,769,948 bp and ending after 96,769,369 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37699 | ATG CTT AGA CAG ACA GCC ATG T | Common | A | |||
| 37700 | CCT CAG ACC TTT GGC TAG TGA | Mutant Reverse | A | |||
| 37701 | ATA CAC AAC GCT GGC ATG AG | Wild type Reverse | A | |||
| 37702 | Fluorophore-1 | TCA GCA ATG GAT ACA GTA ATG AGC | Quencher-1 | MUT Probe | ||
| 37703 | Fluorophore-2 | CAT CTC ACA GGG CTG TTC CT | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37699 | 0.40 uM |
| 37700 | 0.40 uM |
| 37701 | 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.