Stock Number: 003627 Strain Name: B6C3-Tg(HD82Gln)81Dbo/J

Allele: Tg(HD82Gln)81Dbo Protocol Name: Tg(HD82Gln)81Dbo Method: Standard PCR Version: 2.1 Created: 29-July -2008 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 250 bp Internal positive control = 200 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 oIMR1239 - GTG GAT ACC CCC TCC CCC AGC CTA GAC C - Transgene Reverse oIMR1240 - GAA CTT TCA GCT ACC AAG AAA GAC CGT GT - Transgene Forward oIMR8744 - CAA ATG TTG CTT GTC TGG TG - Internal Positive Control Forward oIMR8745 - GTC AGT CGA GTG CAC AGT TT - Internal Positive Control Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.24 - 3.24 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 oIMR1239 0.48 0.80 uM 0.48 20 uM oIMR1240 0.48 0.80 uM 0.48 20 oIMR8744 0.48 0.80 0.48 20 uM oIMR8745 0.48 0.80 uM 0.48 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 35 sec - 3 64 45 sec* *-0.5 C per cycle 4 72 45 sec repeat steps 2-4 for 12 cycles 5 94 35 sec - 6 58 30 sec - 7 72 45 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 003627 B6C3-Tg(HD82Gln)81Dbo/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.