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Stock No: 004605
Protocol 20690: End Point Analysis Assay - Itgb1<tm1Efu>-EP
Version 1.0
Notes
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= 178 bp
Wild Type = 89 bp
Fam=Mut
Hex=Wt
Sequence
SEQ
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 30544 | Fluorophore-1 | CTC GAG GGA CCT AAT AAC TTC GT | Quencher-1 | MUT Probe | ||
| 30888 | AAA CAT TTA ATT TGC GGC TCA | Forward | A | |||
| 30889 | CCA GCT CCA TGT CTG TCT GT | Reverse | A | |||
| 30890 | Fluorophore-2 | TGT CAG TCC ATG GCC GTC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 30888 | 0.40 uM |
| 30889 | 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.