Stock No: 002955
Protocol 23401: Standard PCR Assay - Gt(ROSA)26Sor STD
Version 1.2

Notes

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

Mutant = ~500 bp
Heterozygote = 374 bp and ~500 bp
Wild type = 374 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
oIMR0092 AAT CCA TCT TGT TCA ATG GCC GAT C Mutant A Neo R
oIMR0314 GGC TTA AAG GCT AAC CTG ATG TG Wild type A
oIMR3449 CCG GAT TGA TGG TAG TGG TC Mutant A LacZ F
oIMR8546 GGA GCG GGA GAA ATG GAT ATG Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0092 0.50 uM
oIMR0314 0.50 uM
oIMR3449 0.50 uM
oIMR8546 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
002292 129-Gt(ROSA)26Sor/J
003504 B6;129-Gt(ROSA)26Sortm1Sho/J
002073 B6;129S-Gt(ROSA)26Sor/J
002955 C.129S7-Gt(ROSA)26Sor/J
4 strains use this protocol