For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 023837
Protocol 37832: Probe Assay - Snca<tm1Rosl>-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 = 91 bp
Wt = 94 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence:
gctctctggtgtggcatctgggctcgtcctggaggagggggtcgcctagaggaactgggaacagactgaggcagggaaggaggggggtggggcaggagaggcgccagctcaagttcagccacgataaaactgagggccctctgaacTCGAGGGGAGGCtcaggccgtcctctcttccttccatccgggggaatgtgctccagatacccacagccctcacgcaccgcacctccaaccaacccgtcccctccctaggaagaggagcgaaggcacgaggcaggcgaggggcggggagaggcgctgacaaatcagctgcgggggcgacgtgaaggagccagggagccagagcgcccggcagcaggcagcagacggcaggagaccagcaggtgttcccc
Mutant Sequence:
TCGCCTAGAGGAACTGGGAACAGACTGAGGCAGGGAAGGAGGGGGGTGGGGCAGGAGAGGCGCCAGCTCAAGTTCAGCCACGATAAAACTGAGGGCCCTCTGAACTCGAccgcatgcctgtacactccggaagacgcgttcggccgtgctagcgaattctggatccacgatctagagtcagcttctgatggaattagaacttggcaaaacaatactgagaatgaagtgtatgtggaacagaggctgctgatctcgttcttcaggctatgaaactgacacatttggaaaccacagtacttagaaccacaaagtgggaatcaagagaaaaacaatgatcccacgagagatctatagatctatagatcatgagtgggaggaatgagctggcccttaatttggttttgcttgtttaaattatgatatccaactatgaaacattatcataaagcaatagtaaagagccttcagtaaagagcaggcatttatctaatcccaccccacccccacccccgtagctccaatccttccattcaaaatgtaggtactctgttctcacccttcttaacaaagtatgacaggaaaaacttccattttagtggacatctttattgtttaatagatcatcaatttctgcagacttacagcggatcGATCCCCTCAGAAGAACTCGTCAAGAAGG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52272 | CAG CCA CGA TAA AAC TGA GG | Common | A | |||
| 52273 | TGG GTA TCT GGA GCA CAT TC | Wild type Reverse | A | |||
| 52274 | GAT CCA GAA TTC GCT AGC AC | Mutant Reverse | A | |||
| 52275 | Fluorophore-1 | ACT CGA GGG GAG GCT CAG G | Quencher-1 | WT Probe | ||
| 52276 | Fluorophore-2 | CCT GTA CAC TCC GGA AGA CGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52272 | 0.40 uM |
| 52273 | 0.40 uM |
| 52274 | 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.