Stock Number: 003653 Strain Name: NOD.129X1-Gad2tm1Bae/J

Allele: Gad2tm1Bae Protocol Name: Gad2tm1Bae Method: Standard PCR Version: 2.0 Created: 15-May -2009 Updated: 24-September-2009

Expected Results: Mutant = 530 bp Heterozygote = 315 bp and 530 bp Wild type = 315 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 oIMR2060 - CAC GAG ACT AGT GAG ACG TG - Mutant oIMR3457 - CAT ACG CAG ACA GCA CGT TT - Common oIMR3473 - CAA ACC CTA AAC CAC CCA CA - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.94 - 3.94 10 X High Fidelity Buffer 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 oIMR3457 0.60 1.00 uM 0.60 20 uM oIMR2060 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul High Fidelity Platinum Taq 0.08 0.03 U/ul 0.08 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 53 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 4.26 - 4.26 10 X AB PCR BufferII 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 oIMR3457 0.60 1.00 uM 0.60 20 uM oIMR3473 0.30 0.50 uM 0.30 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.06 0.01 U/ul 0.06 DNA 2.00 - 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 003653 NOD.129X1-Gad2tm1Bae/J 003654 B6.129X1-Gad2tm1Bae/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 30 sec - 3 53 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold