Genotyping Protocols Database
Protocol 29479: Standard PCR Assay - Gt(ROSA)26Sor<tm1(CAG-Brainbow2.1)Cle>
Version 3.2

Notes

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

8916 is the common primer

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 = 300 bp
Heterozygote = 300 bp and 386 bp
Wild type = 386 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11341 GAA TTA ATT CCG GTA TAA CTT CG Mutant Forward A directly after mCerulean
oIMR8545 AAA GTC GCT CTG AGT TGT TAT Wild type Forward A
oIMR8916 CCA GAT GAC TAC CTA TCC TC Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
11341 0.50 uM
oIMR8545 0.50 uM
oIMR8916 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
017492 B6.129P2-Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle/J
013731 STOCK Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle/J
2 strains use this protocol