Stock Number: 002939 Strain Name: B6.129S4-Insrtm1Dac/J

Allele: Insrtm1Dac Protocol Name: Insrtm1Dac Method: Separated PCR Version: 1.1 Created: 04-June -2008 Updated: 24-September-2009

Expected Results: Mutant = 700 bp Heterozygote = 120 bp and 700 bp Wild type = 120 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0234 - CGC CAA GCT CTT CAG CAA TAT - Mutant oIMR0237 - CTT GAT GTG CAC CCC ATG TCT - Wild type oIMR0238 - TCG GAT GTT GAT GAT CAG GCT - Wild type oIMR1535 - GAT CGG CCA TTG AAC AAG ATG - -

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.62 - 3.62 10 X PCR Buffer II (ABI) 1.30 1.18 X 1.30 25 mM MgCl2 1.30 2.96 mM 1.30 2.5 mM dNTP 1.04 0.24 mM 1.04 20 uM oIMR0237 0.52 0.95 uM 0.52 20 uM oIMR0238 0.52 0.95 uM 0.52 5 mM DNA Loading Dye 1.66 0.76 mM 1.66 5 U/ul Taq DNA Polymerase 0.04 0.02 U/ul 0.04 DNA 1.00 - 1.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.62 - 3.62 10 X PCR Buffer II (ABI) 1.30 1.18 X 1.30 25 mM MgCl2 1.30 2.96 mM 1.30 2.5 mM dNTP 1.04 0.24 mM 1.04 20 uM oIMR1535 0.52 0.95 uM 0.52 20 uM oIMR0234 0.52 0.95 uM 0.52 5 mM DNA Loading Dye 1.66 0.76 mM 1.66 5 U/ul Taq DNA Polymerase 0.04 0.02 U/ul 0.04 DNA 1.00 - 1.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 002426 B6;129S4-Insrtm1Dac/J 002939 B6.129S4-Insrtm1Dac/J 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 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold