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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
>chr7:46178283-46178382 100bp GGCCTTCTGTAAACCAGTACC ATCTGCACCCCTTTATCCTC
Mut= 99 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CGTCTCAAAAATAAATAAATAAATAAATAAATAAATAAATAAAGAGCTGCTGCAGAAGGAAGCTGCTAGTTACTGACAGAGGTAAATGATGTGCATGCCCATATGTCTAGCAGAAACGAAACAGAATGGGTAGACAGTCACACAAGAGGTCAGTGTGGTGGTCCCTGCttaggaccggcctgcaggctcaggctttgtgcctggatatccagttcatatgctcatccctggggaaggaggatttgcatggtctccttatcaccatgctgggcctccaggtgggcgtcctgccttctgcgtctctccccacaggatggggcagccagagctccaaggtacacattcaccatagcacctggctccatttccctggccacaacctgcgctggatccttaccttcatgctgctctttgtcctggtgtgtgagatcgctgagggtatcctgtctgatgggtaagtgagctccaggggagtggggaagctgcaggagaccctccctgtgcctgctcatcggccttctgtaaaccagtacctggtgtgaactgcgccaggccttaggcacatatgagacgcAGGGCCTACCATCAGTCTGGAGGATAAAGGGGTGCAGATTTCTGTACCAGTTCCCAGTGAGACCATTGGAGAGCCTTGTCAGAGTGTCCCAGCTGCTGTGGGCAATACCCGGTCTCTCTGAAAGTGACTGAAGTGCGTGAGGGGACAGTAAATCCTGTCACTTGTTCTTACTGAAGGCCTCTCCTTGGTTCCCA
This mutation is a 406 bp deletion beginning at Chromosome 7 position 46,178,322 bp and ending after 46,178,727 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44885 | GGC CTT CTG TAA ACC AGT ACC | Wild type Forward | A | |||
| 44886 | ATC TGC ACC CCT TTA TCC TC | Common | A | |||
| 44887 | AGC AGA AAC GAA ACA GAA TGG | Mutant Forward | A | |||
| 44888 | Fluorophore-1 | AAC TGC GCC AGG CCT TA | Quencher-1 | WT Probe | ||
| 47061 | Fluorophore-2 | AGG TCA GTG TGG TGG TCC C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44885 | 0.40 uM |
| 44886 | 0.40 uM |
| 44887 | 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.