Stock Number: 002720 Strain Name: AKR/CumJ

Allele: Rag1tm1Mom Protocol Name: Rag1tm1Mom Method: Standard PCR Version: 1.1 Created: 09-February -2001 Updated: 24-September-2009

Expected Results: Mutant = 530 bp Heterozygote = 474 bp and 530 bp Wild type = 474 bp

Gel Image

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

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1746 - GAG GTT CCG CTA CGA CTC TG - Wild type Forward oIMR3104 - CCG GAC AAG TTT TTC ATC GT - Common oIMR8162 - TGG ATG TGG AAT GTG TGC GAG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.68 - 3.68 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 oIMR1746 0.60 1.00 uM 0.60 20 uM oIMR3104 0.30 0.50 uM 0.30 20 uM oIMR8162 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 0.04 0.02 U/ul 0.04 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 2 min - 2 94 30 sec - 3 58 45 sec - 4 72 45 sec 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 002096 B6;129S7-Rag1tm1Mom/J 002216 B6.129S7-Rag1tm1Mom/J 002506 STOCK Tg(CD3E)26Cpt-Rag1tm1Mom/J 002720 AKR/CumJ 003145 C.129S7(B6)-Rag1tm1Mom/J 003729 NOD.129S7(B6)-Rag1tm1Mom/J 004259 NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/+ Tg(TcrbAI4)1Dvs/+ 004347 NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ 004348 NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ 004369 B6.Cg-Rag1tm1Mom Ins2Akita/J 004673 NOD.129(B6)-Rag1tm1Mom Cd80tm1Shr/JbsJ 004848 NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ 005273 NOD.Cg-Rag1tm1Mom Cd86tm2Shr Cd80tm1Shr/JbsJ 005707 STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J 006022 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ 006024 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ 006770 STOCK Rag1tm1Mom Tg(TIE2GFP)287Sato/J 007799 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.