Genotyping Protocols Database
Protocol 28710: Standard PCR Assay - Gt(ROSA)26Sor(CAG-EYFP)
Version 3.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.

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

3%

Mutant = 212 bp
Heterozygote = 212 bp and 297 bp
Wild type = 297 bp

Note:  Stock# 012569 and Stock# 024109 have a mutant band size = 253 bp due to different polylinker length between primer annealing sites.

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
oIMR9020 AAG GGA GCT GCA GTG GAG TA Wild type Forward A
oIMR9021 CCG AAA ATC TGT GGG AAG TC Wild type Reverse A
oIMR9102 ACA TGG TCC TGC TGG AGT TC Mutant Forward A YFP/EGFP
oIMR9103 GGC ATT AAA GCA GCG TAT CC Mutant Reverse A WPRE

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR9020 0.50 uM
oIMR9021 0.50 uM
oIMR9102 0.50 uM
oIMR9103 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
007903 B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J
024109 B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J
2 strains use this protocol