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Stock No: 017681
Protocol 30655: Probe Assay - Idua<tm1.1Kmke> 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= 95 bp
Wild Type = 113 bp
>chr5:109110273+109110385 113bp CATCCTGTGGGCTGAACA AGAGGGCTGAGGCTGTTG
Sequence
Wt Sequence:tccaaatttctacctcttcatctcctcccatcctgtgggctgaacagtataacagactcccaGTATACaaatggtgggagctagatattagggtaggaagccagatgctaggtatgagagagccaacagcctcagccctctgcttggcttatagATGGAGAACAACTCtggGCAGAGGTCTCAAAG
Mutant Sequence: catctcctcccaTCCTGTGGGCTGAACAGTATAACAGACTCCCAGTAgggctgcaggaattcgatatcaagcttatcgataccgtcgagggacctaATAACTTCGTATAGCATACATTATACGAAGTTatattaagggttattgaatatgatcggaattcctcgagcggccTACAAATGGTGGGAGCTAGATATTAGGGTAGGAAGCCAGATGCTAGGTATGAGAGAGCCAACAGCCTCAGCCCTCTGCTTGGCTTATAGATGGAGAACAACTCTAGGCAGAGGTCTCAAAGGCTGGGGCTGTGTTGGACAG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 35127 | CAT CCT GTG GGC TGA ACA | Common | A | |||
| 35128 | AGA GGG CTG AGG CTG TTG | Wild type Reverse | A | |||
| 35129 | Fluorophore-1 | CCC AGT AGG GCT GCA GGA A | Quencher-1 | MUT Probe | ||
| 35130 | Fluorophore-2 | ACT CCC AGT ATA CAA ATG GTG GGA | Quencher-2 | WT Probe | ||
| oIMR7882 | CGA AGT TAT TAG GTC CCT CGA C | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 35127 | 0.40 uM |
| 35128 | 0.40 uM |
| oIMR7882 | 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.