Stock Number: 005987 Strain Name: 129-Achetm1Loc/J

Allele: Achetm1Loc Protocol Name: Achetm1Loc Method: Standard PCR Version: 1.1 Created: 23-June -2008 Updated: 24-September-2009

Notes: Optimized using Platinum High Fidelity Taq (Invitrogen) with a corresponding buffer. Expected Results: Mutant = 319 bp Heterozygote = 164 bp and 319 bp Wild type = 164 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 oIMR4266 - AAC ATT GGC CGC CTC CAG CTC - Mutant oIMR4267 - TGG AAG GTG CCA CTC CCA CTG - Mutant oIMR4268 - AAT GAC ACC GAG CTG ATA GCC - Wild type oIMR4269 - CCA GTA TTG ATG AGA GCC TCC G - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.76 - 2.76 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 oIMR4266 0.60 1.00 uM 0.60 20 uM oIMR4267 0.60 1.00 uM 0.60 20 uM oIMR4268 0.60 1.00 uM 0.60 20 uM oIMR4269 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 0.06 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 68 30 sec - 4 72 30 sec 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 005987 129-Achetm1Loc/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.