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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
>chr3:153850467+153850562 96bp ACACAGCAGCGACACACAG AGGAAAGAAAACTGCCACCA
Mut= 113 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence: (deleted region in lower case)
AGTTCACACAGATATAGGCACACGTAACTAAAAAAAATCAAATCTTTAAAAAATAAGCAATAAGAAAACTGTTTTTATTTTTAATTTTCATGTTATATTTGCAGAGCATGGTAAGGTTTATGCCCCCACCCGTGGGGGAGACACAGCAGCGACACACAGCTGCAGCGACTAAGTGCTTTGCCCATGTGGTGTGATGTgaaaggcggcttcagcatttggtggcagttttctttcctccttcagtttggtccacagttacaatgtcaaatccttcttaagacattacaggtgaacgttagttacatggtggtcactctgcttttggcttgcagaaccttcacacctgtgtactgccccagccccctgagtggcatcacacctctactctacgtggctcagacaagacagtcaaatatcttaaaaattctcctgcagtatggaatcctagaaagagaaaaaaaccctatcaacattgttctgacaatactactttacccttcgagagtgagaataatggttgaccacgagttgattgacattcaagaagatgccaagacatgtttaatgctatgttccagagtgctttctacgatctcagtcagggagatagaggtaagcggagcaccgcccttctcagggcgtggggagtggataataagagaagcagaggaacacttttggggtttaagcagagctatattctatggggaaaagccgagtgttgctctttggggtgcaatttaccatcagtaagtgttaggacataattgcaaggGAAAATTGCAGAGTATTTTTAATTTATGCATTATTTCCAATGGTGAATTAATGACCTCGCATATGCTCAGTAATTTAAAATGAGGCAAAATTCAAATTAACTAAAATTAGTCCTTCAAATGTTGATTTTTTTTGTTACCGTGAACTTCAAAATGGCAAAGCTGAGTGTCCTATAGACA
This mutation is a 578 bp deletion beginning at Chromosome 3 positive strand position 153,850,524 bp and ending after 153,851,101 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38437 | ACA CAG CAG CGA CAC ACA G | Common | A | |||
| 38438 | AGG AAA GAA AAC TGC CAC CA | Wild type Reverse | A | |||
| 38439 | GGT CAT TAA TTC ACC ATT GGA | Mutant Reverse | A | |||
| 38440 | Fluorophore-1 | AAA GGC GGC TTC AGC ATT | Quencher-1 | WT Probe | ||
| 39176 | Fluorophore-2 | TGT GAT GTG AAA ATT GCA GAG TAT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38437 | 0.40 uM |
| 38438 | 0.40 uM |
| 38439 | 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.