Stock Number: 002082 Strain Name: 129S-Rb1tm1Tyj/J

Allele: Rb1tm1Tyj Protocol Name: Rb1tm1Tyj Method: Standard PCR Version: 1.1 Created: 13-August -2008 Updated: 24-September-2009

Expected Results: Mutant = ~370 bp Heterozygote = 310 bp and ~370 bp Wild type = 310 bp

Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0025 - AAT TGC GGC CGC ATC TGC ATC TTT ATC GC - Forward oIMR0026 - CCC ATG TTC GGT CCC TAG - Reverse oIMR0027 - GAA GAA CGA GAT CAG CAG - -

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR0025 0.60 1.00 uM 0.60 20 uM oIMR0026 0.60 1.00 uM 0.60 20 uM oIMR0027 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 60 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 002082 129S-Rb1tm1Tyj/J 002102 B6.129S2-Rb1tm1Tyj/J 002546 C3Ou.129S2-Rb1tm1Tyj/J 002900 FVB.129S2(B6)-Rb1tm1Tyj/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.