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Stock No: 027929
Protocol 30590: Probe Assay - Yap1<tm1.1Dupa> Probe Alternate2
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= 108 bp
Wild Type = 156 bp
>chr9:8007494-8007649 156bp GGTGGATTTCTCAGTTCAAGG AGTCAGGGTTTCCATTGCTG
Sequence
Wt Sequence: taggctttaagaccctcctctagccgggcgtggtggcacacgcctctaatcccagcacttgggagacagaggtggatttctcagttcaaggccagccttggtccaggaccaggacagccaggactacacagagaacccTGTctggaaaaaaccaaaaaaaaaaaaaaaaaaagagagagagttgttttggtcatggtgtctctccacagcaatggaaaccctgactaagacaaatggtaagggagagtcttgatccagttatcatagcaagtgtgttctcaatttaaaggctggtgaaacaatattgaccaaagcacacAAGcccattaattgacagtgcccaatctagtaga
Mutant Sequence: TTGGGAGACAGAGGTGGATTTCTCAGTTCAAGGCCAGCCTTGGTCCAGGACCAGGACAGCCAGGACTACACAGAGAACCCTGaattcctgcagcccaattccgatcatattcaataacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcgaggggatccgTCTGGAAAAAACCAAAAAAAAAAAaaaaaaaagagagagagttgttttggtcatggtgtctctccacagcaatggaaaccctgactaagacaaatggtaagg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 33558 | GGT GGA TTT CTC AGT TCA AGG | Common | A | |||
| 33559 | AGT CAG GGT TTC CAT TGC TG | Wild type Reverse | A | |||
| 33562 | Fluorophore-1 | TGT TTT GGT CAT GGT GTC TCT C | Quencher-1 | WT Probe | ||
| 34563 | Fluorophore-2 | AAC CCT GAA TTC CTG CAG C | Quencher-2 | MUT Probe | ||
| 34954 | GGG TTA TTG AAT ATG ATC GGA ATT G | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 33558 | 0.40 uM |
| 33559 | 0.40 uM |
| 34954 | 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.