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Stock No: 030320
Protocol 31544: Probe Assay - Abca7 Founder 17-3 (KI + 33bp del) - EP
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= 96 bp
Wt= 129 bp
Sequence
TATCTGAAG(c/g)TacactgtgagtccatccatttactatcagtggTCATGTTGCTCA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37121 | TGA ACT TGA CCA AGG AGC AG | Forward | A | |||
| 37122 | TTT GTC ACT AGC CCT GAG CA | Reverse | A | |||
| 37123 | Fluorophore-1 | AGC TAC ACT GTG AGT CCA TCC A | Quencher-1 | WT Probe | ||
| 37124 | Fluorophore-2 | TGA AGG TCA TGT TGC TCA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37121 | 0.40 uM |
| 37122 | 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.