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Stock No: 024574
Protocol 35132: End Point Analysis Assay - Gba1<tm1.1Eginn>-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
Mut = CC
WT= TT
Sequence
gagtctggctgtgttgcccagggaagcctggggtcctgatgatttctatcttcacgttcagCAAGTTCATTCCCGAGGGATCCCAGAGAGTGGCGTTGGTGGCCAGTGAGAGCACTGAC(TT/CC)GGAAACAGTAGCACTGTTACGCCCTGACGGCTCTGCAGTTGTGGTCGTGTTAAACcGgtaagctgggtgcaagggcaggtct
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45337 | ATC CCA GAG AGT GGC GTT G | Forward | A | |||
| 45338 | Fluorophore-1 | AGA GCA CTG ACT TGG AAA CAG TAG | Quencher-1 | WT Probe | ||
| 45339 | Fluorophore-2 | AGC ACT GAC CCG GAA ACA GTA | Quencher-2 | MUT Probe | ||
| oIMR1804 | ACA CGA CCA CAA CTG CAG AG | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45337 | 0.40 uM |
| oIMR1804 | 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.