Genotyping Protocols Database
Protocol 29436: Standard PCR Assay - Gt(ROSA)26Sor(tdTomato-WPRE)
Version 2.2

Notes

Melt 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

Mutant Tm = 81°C +/- 1.0°C
Wild type Tm = 87°C +/- 1.0°C

Mutant  = 196 bp
Heterozygote = 297 bp and 196 bp
Wild type = 297 bp

For Stock# 012567
Mutant = ~315 bp
Wild type = 297 bp
 
For Stock #7914
Mutant = ~200 bp
Wild type = 297 bp

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
oIMR9103 GGC ATT AAA GCA GCG TAT CC Mutant Reverse A WPRE
oIMR9105 CTG TTC CTG TAC GGC ATG G Mutant Forward A tdTomato

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
oIMR9103 0.50 uM
oIMR9105 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
007905 B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J
007908 B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J
007909 B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J
007914 B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J
012567 B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hze/J
012570 B6;129S-Gt(ROSA)26Sortm34.1(CAG-Syp/tdTomato)Hze/J
021875 B6;129S-Gt(ROSA)26Sortm65.1(CAG-tdTomato)Hze/J
021876 B6;129S-Gt(ROSA)26Sortm66.1(CAG-tdTomato)Hze/J
025106 B6.Cg-Gt(ROSA)26Sortm75.1(CAG-tdTomato*)Hze/J
030855 STOCK Notch1tm2Rko Notch2tm3Grid Gt(ROSA)26Sortm9(CAG-tdTomato)Hze Tg(KRT5-cre/ERT2)2Ipc/JeswJ
031800 B6.Cg-Gad2tm1Rpa Gad1tm1Rpa Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/RpaJ
032864 B6.Cg-Gt(ROSA)26Sortm65.2(CAG-tdTomato)Hze/J
12 strains use this protocol