Stock Number: 004133 Strain Name: B6.129S7-Apptm1Dbo/J

Allele: Apptm1Dbo Protocol Name: Apptm1Dbo Method: Standard PCR Version: 1.1 Created: 30-April -2003 Updated: 02-November -2009

Expected Results: Mutant = 280 bp Heterozygote = 161 bp and 280 bp Wild type = 161 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 oIMR1636 - AGA GCA CCG GGA GCA GAG - Wild type oIMR1637 - AGC AGG AGC AGT GCC AAG - Wild type oIMR6916 - CTT GGG TGG AGA GGC TAT TC - Mutant Forward oIMR6917 - AGG TGA GAT GAC AGG AGA TC - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.06 - 3.06 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 oIMR6916 0.30 0.50 uM 0.30 20 uM oIMR6917 0.30 0.50 uM 0.30 20 uM oIMR1636 0.70 1.17 uM 0.70 20 uM oIMR1637 0.70 1.17 uM 0.70 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.08 0.01 U/ul 0.08 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.08 0.01 U/ul 0.08 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 64 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 004133 B6.129S7-Apptm1Dbo/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.