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 --
1 95.0 --
2 95.0 --
2 95.0 --
3 60.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
4 -- repeat steps 2-3 for 40 cycles
5 4.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
024338 B6.Cg-Sncatm1Rosl Tg(SNCA*A30P)192Rwm/J
023837 B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J
003692 B6;129X1-Sncatm1Rosl/J
3 strains use this protocol