Stock Number: 004598 Strain Name: B6;129S4-Gt(ROSA)26Sortm2Dym/J

Allele: Gt(ROSA)26Sortm2Dym Protocol Name: Gt(ROSA)26Sortm2Dym Method: Standard PCR Version: 2.1 Created: 29-August -2008 Updated: 24-September-2009

Expected Results: Mutant = 630 bp Heterozygote = 500 bp and 630 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 oIMR1427 - TGT TTT GGA GGC AGG AAG CAC TTG - Wild type oIMR1428 - AAA TAC TCC GAG GCG GAT CAC AAG - Wild type oIMR2028 - GCC GTC CTT GAG CAC ATA G - Mutant oIMR2029 - GGA CAT TGA CGT GAT CCT AG - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 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 0.30 0.50 uM 0.30 20 uM oIMR1428 0.30 0.50 uM 0.30 20 uM oIMR2028 0.60 1.00 uM 0.60 20 uM oIMR2029 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 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 63 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 004598 B6;129S4-Gt(ROSA)26Sortm2Dym/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.