Genotyping Protocols Database
Stock No: 004435
Protocol 24173: Standard PCR Assay - Tg(SOD1)
Version 2.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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 = 236 bp
Internal positive standard = 324 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0113 CAT CAG CCC TAA TCC ATC TGA Transgene Forward A SOD1
oIMR0114 CGC GAC TAA CAA TCA AAG TGA Transgene Reverse A SOD1
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C 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
oIMR0113 0.50 uM
oIMR0114 0.50 uM
oIMR7338 0.50 uM
oIMR7339 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
002297 B6SJL-Tg(SOD1)2Gur/J
002300 B6SJL-Tg(SOD1*G93A)dl1Gur/J
002628 C57BL/6-Tg(SOD1)10Cje/J
002726 B6SJL-Tg(SOD1*G93A)1Gur/J
004435 B6.Cg-Tg(SOD1*G93A)1Gur/J
008248 B6.Cg-Tg(SOD1*G85R)148Dwc/J
008342 B6.Cg-Tg(SOD1*G37R)42Dpr/J
7 strains use this protocol