Stock Number: 006999 Strain Name: STOCK Dbttm1Geh Tg(tTALap)5Bjd Tg(tetO-DBT)A1Geh/J

Allele: Dbttm1Geh Protocol Name: Dbttm1Geh Method: Separated PCR Version: 1.1 Created: 03-July -2008 Updated: 24-September-2009

Expected Results: Mutant = 500 bp Heterozygote = 210 bp and 500 bp Wild type = 210 bp

Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR2060 - CAC GAG ACT AGT GAG ACG TG - Mutant oIMR6993 - GGC TGC ACT GTT ACA AGC AT - Wild type Forward oIMR6994 - GTG CTG CAT TCA CAA TGA CC - Common

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 oIMR6994 0.60 1.00 uM 0.60 20 uM oIMR2060 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 60 1 min - 4 72 1.5 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

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 oIMR6993 0.60 1.00 uM 0.60 20 uM oIMR6994 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 This genotyping protocol is used for the following strains:   Stock Number Strain Name 006999 STOCK Dbttm1Geh Tg(tTALap)5Bjd Tg(tetO-DBT)A1Geh/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 60 1 min - 4 72 1.5 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold