Stock Number: 008191 Strain Name: B6;129S2-Trp53tm1Tyj Nf1tm1Tyj/J

Allele: Trp53tm1Tyj Protocol Name: Trp53tm1Tyj Method: Standard PCR Version: 2.1 Created: 25-September-2007 Updated: 24-September-2009

Notes: This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase). Expected Results: Mutant = 600 bp Heterozygote = 400 bp and 600 bp Wild type = 400 bp

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7777 - ACA GCG TGG TGG TAC CTT AT - Wild type Forward oIMR7778 - TAT ACT CAG AGC CGG CCT - Wild type Reverse oIMR8306 - CTA TCA GGA CAT AGC GTT GG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.67 - 3.67 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 oIMR7777 0.05 0.08 uM 0.05 20 uM oIMR7778 0.60 1.00 uM 0.60 20 uM oIMR8306 0.70 1.17 uM 0.70 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.20 0.02 U/ul 0.20 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 1.5 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 002080 129-Trp53tm1Tyj/J 002101 B6.129S2-Trp53tm1Tyj/J 002103 B6;129S2-Trp53tm1Tyj/J 002526 C.129S2(B6)-Trp53tm1Tyj/J 002547 C3Ou.129S2(B6)-Trp53tm1Tyj/J 002899 FVB.129S2(B6)-Trp53tm1Tyj/J 008191 B6;129S2-Trp53tm1Tyj Nf1tm1Tyj/J 302526 C.129S2(B6)-Trp53tm1Tyi/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.