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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
>chr3:95790391-95790529 139bp TTAAGGGTTTTTCGGTGCAT AACTGAAAAGCAGCCAGTGC
Mut= 139 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CCATTTTTTAATTGGGTTGTTTGTTTTTTTGGTGGTTAGCTTCTTGTTTTAATTTCTTTAATGGATTTTTTTTTTCTTTAGTGCCAAGTGTCTACAGAATGAGGTAGTCTACTTGGAAGAGTACCTTGAACACAAATTTAACCTCAAATAGTTAAGGGTTTTTCGGTGCATTTAATAAATTATCCATTAGttaatttcttattaccccctccatgtaattagtccatgtaaaagcaattacgtgaggctggagagatgctcaggttggaagcactggctgcttttcagttcctttctggcctccgtaggcactgcatgtgggtgatgtgcatacaagcaggcaaggcacccataagcacaaagtaacagcatggaggtttttaaaagttagctgtttgaaaacagtatatttgggaaaagttaatgacttttttttatctcttctctccagctacatatccccatcgtttgaatctcttttatcttgccaatgatgtcatacagaattgtaaacggaaaaatgcaatcatatttcgtgaatcatttgctgatgtgcttcctgaagcagctgctctagtgaagtaagtaaatctgtattgtacttggggattaattttttaatttttattttcttgtttttgcacttattaacaaaaacataaattgtgcatatttaaagacccaagtaaaatattgtgttattggacaagtgTAATGGTGTTTGTTTTTAATTTCAGCATCTGGGGAGCGGGGAAGGTGAAACTTTTAAATTTGAGACCTCGTTTCAAAAAAGCAAAAATGGGGTGAGCATAGGAGGTAGTAGCTCATTCTTATCTTTCTCTGGATTCATTCATAGTTTAATTTTTAGTGTTC
This mutation is a 522 bp deletion beginning at Chromosome 3 position 95,789,969 bp and ending after 95,790,490 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41629 | TTA AGG GTT TTT CGG TGC AT | Common | A | |||
| 41630 | AAC TGA AAA GCA GCC AGT GC | Wild type Reverse | A | |||
| 41631 | TAT GCT CAC CCC ATT TTT GC | Mutant Reverse | A | |||
| 41632 | Fluorophore-1 | AGC AAT TAC GTG AGG CTG GA | Quencher-1 | WT Probe | ||
| 41633 | Fluorophore-2 | ATC CAT TAG TAA TGG TGT TTG TTT TTA A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41629 | 0.40 uM |
| 41630 | 0.40 uM |
| 41631 | 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.