Stock Number: 000285 Strain Name: B6.Cg-Rorasg + +/+ Myo5ad Bmp5se/J

Allele: Rorasg Protocol Name: Rorasg Method: Standard PCR Version: 1.1 Created: 25-August -2000 Updated: 24-September-2009

Expected Results: Mutant = 450 bp Heterozygote = 318 bp and 450 bp Wild type = 318 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 oIMR1233 - TCT CCC TTC TCA GTC CTG ACA - Wild type oIMR1234 - TAT ATT CCA CCA CAC GGC AA - Wild type oIMR1235 - GAT TGA AAG CTG ACT CGT TCC - Mutant oIMR1236 - CGT TTG GCA AAC TCC ACC - 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 oIMR1233 0.45 0.75 uM 0.45 20 uM oIMR1234 0.45 0.75 uM 0.45 20 uM oIMR1235 0.45 0.75 uM 0.45 20 uM oIMR1236 0.45 0.75 uM 0.45 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 54 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 000237 B6C3Fe a/a-Rorasg/J 000285 B6.Cg-Rorasg + +/+ Myo5ad Bmp5se/J 002651 B6.C3(Cg)-Rorasg/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.