Stock Number: 009086 Strain Name: B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ

Allele: Gt(ROSA)26Sortm1(FLP1)Dym Protocol Name: Gt(ROSA)26Sortm1(FLP1)Dym Method: Separated PCR Version: 1.1 Created: 03-February -2004 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 = 725 bp Heterozygote = 500 bp and 725 bp Wild type = 500 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 oIMR1348 - CAC TGA TAT TGT AAG TAG TTT GC - Transgene oIMR1349 - CTA GTG CGA AGT AGT GAT CAG G - Transgene oIMR1427 - TGT TTT GGA GGC AGG AAG CAC TTG - Wild type oIMR1428 - AAA TAC TCC GAG GCG GAT CAC AAG - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.42 - 3.42 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 oIMR1348 0.80 1.33 uM 0.80 20 uM oIMR1349 0.80 1.33 uM 0.80 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 64.6 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.42 - 3.42 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 oIMR1427 0.80 1.33 uM 0.80 20 uM oIMR1428 0.80 1.33 uM 0.80 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 This genotyping protocol is used for the following strains:   Stock Number Strain Name 003946 129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J 009086 B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 64.6 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold