Stock Number: 008204 Strain Name: B6;129S4-Pou5f1tm1Jae/J

Allele: Pou5f1tm1Jae Protocol Name: Pou5f1tm1Jae Method: Separated PCR Version: 1.1 Created: 11-July -2008 Updated: 24-September-2009

Expected Results: Mutant = ~450 bp Heterozygote = 249 bp and ~450 bp Wild type = 249 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 oIMR6218 - CCT TCT ATC GCC TTC TTG ACG - Mutant oIMR8137 - AAC CTG GAG CTT TGG GGT TA - Wild type Forward oIMR8138 - GAT CAC CTG GGG TTT GAG AA - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 4.20 - 4.20 10 X PCR Buffer II (ABI) 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR8138 0.60 1.00 uM 0.60 20 uM oIMR6218 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 5 min - 2 94 30 sec - 3 61 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 4.20 - 4.20 10 X PCR Buffer II (ABI) 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR8137 0.60 1.00 uM 0.60 20 uM oIMR8138 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 This genotyping protocol is used for the following strains:   Stock Number Strain Name 008204 B6;129S4-Pou5f1tm1Jae/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 5 min - 2 94 30 sec - 3 61 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**