Stock Number: 008659 Strain Name: NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ

Allele: Prf1tm1Sdz Protocol Name: Prf1tm1Sdz Method: Standard PCR Version: 1.1 Created: 05-September-2000 Updated: 24-September-2009

Expected Results: Mutant = 250 bp Heterozygote = 250 bp and 187 bp Wild type = 187 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 oIMR1100 - GCT ATC AGG ACA TAG CGT TGG - Mutant oIMR3108 - GGA GGC TCT GAG ACA GGC TA - Reverse oIMR3109 - TAC CAC CAA ATG GGC CAA G - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.38 - 3.38 10 X AB PCR BufferII 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 oIMR3108 0.60 1.00 uM 0.60 20 uM oIMR3109 0.60 1.00 uM 0.60 20 uM oIMR1100 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 3 min - 2 94 30 sec - 3 66 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 002407 C57BL/6-Prf1tm1Sdz/J 003505 NOD.B6-Prf1tm1Sdz/J 004848 NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ 007079 CByJ.B6-Prf1tm1Sdz/J 008659 NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ 014568 NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.