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Stock No: 010911
Protocol 31932: Probe Assay - Wt1<tm1(EGFP/cre)Wtp> 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 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= 89 bp
Wild Type = 110 bp
>chr2:104967440+104967549 110bp CACTTCTCGGGCCAGTTC GCATTGGGGAACATCCTG
Sequence
Wt Sequence: CAAGTCCAGCGCCGAGAATCCGCAGGATCGCAGGAGCGGAGAACCGTCCGCATCCGagccgcaccTCATGGGTTCCGACGTGCGGGACCTGAACGCGCTGCTGCCCGCTGTGTCTTCGCTGGGCGGCGGCGGCGGCGGCTGCGGGCTCCCTGTGAGCGGCGCAGCGCAGTGGGCGCCCGTGTTGGACTTCGCGCCTCCGGGCGCCTCGGCTTACGGGTCGCTGGGCGGTCCCGCGCCTCCTCCCGCTCCGCCGCCGCCTCCGCCGCCACCCCACTCCTTCATCAAACAGGAGCCCAGCTGGGGCGGCGCCGAGCCACACGAGGAGCAGTGCCTGAGCGCCTTCACCTTGCACTTCTCGGGCCAGTTCACCGGTACAGCCGGGGCCTGTCGCTACGGACCCTTCGGTCCTCCCC
Mutant Sequence:GGGCCAAGTCCAGCGCCGAGAATCCGCAGGATCGCAGGAGCGGAGAACCGTCCGCATCCGAGCCGCACCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 13772 | CAC CCC GGT GAA CAG CTC | Mutant Reverse | A | GFP | ||
| 38007 | CGA GAA TCC GCA GGA TCG | Mutant Forward | A | Wt1 | ||
| 38009 | CAC TTC TCG GGC CAG TTC | Wild type Forward | A | |||
| 38010 | Fluorophore-1 | CGC ACC ATG GTG AGC AAG G | Quencher-1 | MUT Probe | ||
| 38011 | Fluorophore-2 | CTG TCG CTA CGG ACC CTT CG | Quencher-2 | WT Probe | ||
| oIMR0151 | GCA TTG GGG AAC ATC CTG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 13772 | 0.40 uM |
| 38007 | 0.40 uM |
| 38009 | 0.40 uM |
| oIMR0151 | 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.