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Stock No: 030304
Protocol 32405: Probe Assay - Il1rap KO (387 bp del) - 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
Mut= 116 bp
Wt= 116 bp
Sequence
Wt Sequence (deletions in lower case):
CAAATTTGTTGTTGGTTCTGACAATAAAGCTTGGGAATATTctaaaacccttggaatggttcttagtctatagttatcacacattcacggtgattattattagaactcttcagggaagaagtaccctctcttactttttaaatttatgttgacaggatgggacttctgtggtatttgatgagtctgtccttctatgggatcctgcagagtcatgcttcgggtgagtatgtggtgctggataaatgactgaaattaatttaacatacaggatcctatatagtgaatttaaatgagcataatgttggttttgttccacagaattctgtagttgcatagaatcaagtgtctgaaagaaccatagaattaggggatgagagaaactagacaacttaggaaggggtctgatgagtataaacacagggacatctCTTCAACTTCATGCCCCACAGGCTAATTCTCTGTATTCTCATCTCAAGTACTGTGGAGGAAATGGCAGGGAGATT
387 bp deletion
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39019 | CAA ATT TGT TGT TGG TTC TGA CA | Common | A | |||
| 39020 | AAT CTC CCT GCC ATT TCC TC | Mutant Reverse | A | |||
| 39021 | Fluorophore-1 | TGG GAA TAT TCT TCA ACT TCA TGC | Quencher-1 | MUT Probe | ||
| 39022 | TCC CTG AAG AGT TCT AAT AAT AAT CAC | Wild type Reverse | A | |||
| 39023 | Fluorophore-2 | TGG GAA TAT TCT AAA ACC CTT GG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39019 | 0.40 uM |
| 39020 | 0.40 uM |
| 39022 | 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.