Stock Number: 005643 Strain Name: B6.129X-Gusbtm1Sly/J

Allele: Gusbtm1Sly Protocol Name: Gusbtm1Sly Method: Restriction Enzyme Digest Version: 1.1 Created: 08-July -2008 Updated: 24-September-2009

Expected Results: Mutant (cut) = 448 bp and 217 bp Heterozygote = 217 bp and 448 bp and 665 bp Wild type (uncut) = 665 bp

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

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR3956 - CCT GTG TCA TTT GCA TGT GAC TAT T - - oIMR3957 - TGT GGG TGC TGG GAA CCA GAC TGA G - -

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 oIMR3956 0.60 1.00 uM 0.60 20 uM oIMR3957 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 1.5 min - 2 94 30 sec - 3 63 30 sec - 4 72 30 sec repeat steps 2-4 for 40 cycles 5 72 2 min - 6 10 - hold**

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 10 X Buffer 1.32 - 1.32 ddH2O 5.68 - 5.68 BstuI 1.00 - 1.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 005643 B6.129X-Gusbtm1Sly/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.