Stock Number: 004446 Strain Name: 129-Lta4htm1Bhk/J

Allele: Lta4htm1Bhk Protocol Name: Lta4htm1Bhk Method: Standard PCR Version: 1.1 Created: 07-July -2003 Updated: 12-November -2009

Expected Results: Mutant = 280 bp Heterozygote = 128 bp and 280 bp Wild type = 128 bp

Gel Image

Image Image file View Full Size

Gel Information: Separated by gel electrophoresis on a 3.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1720 - CGA ATC CAT GCT TAA AAT TGC - Wild type oIMR1721 - GCG TTA CGA ACG TGA GAC AA - 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 3.96 - 3.96 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 oIMR1720 0.30 0.50 uM 0.30 20 uM oIMR1721 0.30 0.50 uM 0.30 20 uM oIMR6916 0.30 0.50 uM 0.30 20 uM oIMR6917 0.30 0.50 uM 0.30 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 60 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 004446 129-Lta4htm1Bhk/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.