Stock Number: 005005 Strain Name: ZRDCT Rax+/ChUmdJ

Allele: Rax+ Protocol Name: Raxey Method: Restriction Enzyme Digest Version: 1.1 Created: 14-April -2009 Updated: 24-September-2009

Expected Results: Mutant = uncut 204 bp Wild type = cuts to 114 bp and 90 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR3583 - GTG GAG GAG GAG AAG GGA AC - - oIMR3584 - GAT GCC GTC TTC CTT GGT AA - -

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.96 - 3.96 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 oIMR3583 0.60 1.00 uM 0.60 20 uM oIMR3584 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 60 30 sec - 4 72 30 sec 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) 10 X AB PCR BufferII 1.20 - 1.20 ddH2O 6.30 - 6.30 Ncol 0.50 - 0.50 This genotyping protocol is used for the following strains:   Stock Number Strain Name 005005 ZRDCT Rax+/ChUmdJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.