Stock Number: 004197 Strain Name: B6.129S6-Rac2tm1Mddw/J

Allele: Rac2tm1Mddw Protocol Name: Rac2tm1Mddw Method: Standard PCR Version: 1.1 Created: 30-October -2003 Updated: 24-September-2009

Expected Results: Mutant = 154 bp Heterozygote = 210 bp and 154 bp Wild type = 210 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 oIMR1549 - GAC GCA TGC TCC ACC CCC T - Common oIMR1550 - TGC CAA GTT CTA ATT CCA TCA GAA GC - Mutant Reverse oIMR1551 - CAC ACA CTT GAT GGC CTG CAT - Wild type Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.59 - 3.59 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.48 0.10 mM 0.48 20 uM oIMR1549 0.60 1.00 uM 0.60 20 uM oIMR1550 0.60 1.00 uM 0.60 20 uM oIMR1551 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 0.07 0.03 U/ul 0.07 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 004197 B6.129S6-Rac2tm1Mddw/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.