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Stock No: 027905
Protocol 30023: Probe Assay - Cd274<tm1Shr> Probe
Version 1.0
Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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= 123 bp
Wild Type = 139 bp
>chr19:29448299+29448437 139bp ACAGACGTCAAGCTGCAGGA GCTGTTTTAAATACGGATGAAGC
Sequence
Wt Sequence: GAGGACCTTAAGCCTCAGCACAGCAACTTCAGGGGGAGAGCCTCGCTGCCAAAGGACCAGCTTTTGAAGGGAAATGCTGCCCTTCAGATCACAGACGTCAAGCTGCAGGACGCAGGCGTTTACTGCTGCATAATCAGCTACGGTGGTGCGGACTACAAGCGAATCACGCTGAAAGTCAATGgtaagaattACcctggatggggaaggcttcatccgtatttaaaacagctccctaatgttgagagctcttcattcttgagagttcgcacgcacttctcacagaacaacagcagcctgttcttctcgctc
Mutant Sequence:tgacgagttcttctgaggggatcggcaataaaaagacagaataaaacgcacgggtgttgggtcgtttgttcggatccgaattcctcgagggcgcgccatttaatggccagcgaggccggtacccaattcgccctatagCCCTGGATGGGGAAGGCTTCATCCGTATTTAAAACAGCTCCCTAATGTTGAGAGCTCTTCATTCTTGAGAGTTCGCACGCACTTCTCACAGAACAACAGCAGCCTGTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 33827 | ACA GAC GTC AAG CTG CAG GA | Wild type Forward | A | |||
| 33828 | GCT GTT TTA AAT ACG GAT GAA GC | Common | A | |||
| 33829 | Fluorophore-1 | CGT TTA CTG CTG CAT AAT CAG CT | Quencher-1 | WT Probe | ||
| 33830 | Fluorophore-2 | ATC CGA ATT CCT CGA GGG | Quencher-2 | MUT Probe | ||
| oIMR5241 | GTG TTG GGT CGT TTG TTC G | Mutant Forward | A | Neo |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 33827 | 0.40 uM |
| 33828 | 0.40 uM |
| oIMR5241 | 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.