Stock Number: 003597 Strain Name: B6.129-Htttm4Mem/J

Allele: Htttm4Mem Protocol Name: Htttm4Mem Method: Separated PCR Version: 1.1 Created: 16-July -2003 Updated: 07-June -2010

Expected Results: Mutant = 677 bp Heterozygote = 424 bp and 677 bp Wild type = 424 bp

Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.**

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1610 - GTC CTG ACA TCG GGA AAG AG - Common oIMR1611 - GTT CCT CTG CCG GAC CTG - Wild type oIMR6533 - GGC TGA GGA AGC TGA GGA G - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 6.14 - 6.14 10 X AM 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 oIMR1610 0.24 0.40 uM 0.24 20 uM oIMR1611 0.24 0.40 uM 0.24 DMSO 1.20 - 1.20 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 1.00 - 1.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 58 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 6.14 - 6.14 10 X AM 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 oIMR1610 0.24 0.40 uM 0.24 20 uM oIMR6533 0.24 0.40 uM 0.24 DMSO 1.20 - 1.20 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 1.00 - 1.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 003455 STOCK Htttm4Mem/J 003597 B6.129-Htttm4Mem/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 58 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**