Stock Number: 003752 Strain Name: C57BL/10ScNJ

Allele: Tlr4lps-del Protocol Name: Tlr4lps-del Method: Standard PCR Version: 1.1 Created: 08-December -2008 Updated: 24-September-2009

Expected Results: Mutant = 140 bp Heterozygote = 140 bp and 390 bp Wild type = 390 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 oIMR8365 - GCA AGT TTC TAT ATG CAT TCT C - Mutant Forward oIMR8366 - CCT CCA TTT CCA ATA GGT AG - Mutant Reverse oIMR8367 - ATA TGC ATG ATC AAC ACC ACA G - Wild type Forward oIMR8368 - TTT CCA TTG CTG CCC TAT AG - Wild type Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.56 - 3.56 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 oIMR8365 0.20 0.33 uM 0.20 20 uM oIMR8366 0.20 0.33 uM 0.20 20 uM oIMR8367 0.60 1.00 uM 0.60 20 uM oIMR8368 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 0.06 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 55 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 003752 C57BL/10ScNJ 005315 B6;C3H-Tg(Scgb1a1-Scnn1b)6608Bouc/J 007227 B6.B10ScN-Tlr4lps-del/JthJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.