Protocol 29811: Standard PCR Assay - Gt(ROSA)26Sor<tm(CAG)>
Version 2.2

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 = 320bp
Heterozygote = 320bp and 603bp
Wild type = 603bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9655 CCA GGC GGG CCA TTT ACC GTA AG Mutant A CAG
oIMR8545 AAA GTC GCT CTG AGT TGT TAT Common A
oIMR8546 GGA GCG GGA GAA ATG GAT ATG Wild type A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
9655 0.50 uM
oIMR8545 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
023035 B6;129S6-Gt(ROSA)26Sortm1(CAG-tdTomato*,-EGFP*)Ees/J
023537 B6N.129S6-Gt(ROSA)26Sortm1(CAG-tdTomato*,-EGFP*)Ees/J
010675 STOCK Gt(ROSA)26Sortm1(CAG-EGFP)Fsh/Mmjax
010701 STOCK Gt(ROSA)26Sortm1.1(CAG-EGFP)Fsh/Mmjax
4 strains use this protocol