Stock No: 008132
Protocol 23862: Standard PCR Assay - Tg(THY1-SNCA*A53T)M53Sud, F53Sud
Version 4.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

Melting curve analysis is done using a Roche Light Cycler 480.
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

Transgene Tm = 86.5°C +/- 1°C
Internal positive control Tm = 83°C +/- 1°C

Transgene = 500 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8584 GGC ACC TAG AGG ATC TCG ACT AGT GG Transgene Forward A
oIMR8585 GGA CCT CGA CGC TTA GGC TTC AGG Transgene Reverse A
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8584 0.50 uM
oIMR8585 0.50 uM
oIMR8744 0.50 uM
oIMR8745 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
008859 B6.Cg-Tg(THY1-SNCA*A53T)F53Sud/J
008135 B6.Cg-Tg(THY1-SNCA*A53T)M53Sud/J
008473 B6;SJL-Tg(THY1-SNCA*A30P)M30Sud/J
008132 STOCK Tg(THY1-Snca)M1mSud/J
4 strains use this protocol