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

Allele: Tg(tetO-DBT)A1Geh Protocol Name: Tg(tetO-DBT)A1Geh Method: QPCR Version: 2.1 Created: 23-July -2008 Updated: 24-September-2009

Notes: Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. Use an appropriate instrument-specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing δCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1544 - CAC GTG GGC TCC AGC ATT - Internal Positive Control Forward oIMR3580 - TCA CCA GTC ATT TCT GCC TTT G - Internal Positive Control Reverse oIMR7738 - ACT GAA CTG AAC CGC CTC CAG AAA - Transgene Forward oIMR7739 - GGT GGC ATT ATC ACT GGT TTG GCA - Transgene Reverse TmoIMR0107 JOE NHS Ester CCA ATG GTC GGG CAC TGC TCA A Black Hole Quencher 1 Probe TmoIMR0111 6-FAM TGA GTC AGC TCA GCA CCA CTG ATC TT Black Hole Quencher 1 Transgene

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 2 X Universal Master Mix 12.50 1.00 X 12.50 40 uM oIMR7738 0.25 0.40 uM 0.25 40 uM oIMR7739 0.25 0.40 uM 0.25 40 uM oIMR1544 0.25 0.40 uM 0.25 40 uM oIMR3580 0.25 0.40 uM 0.25 5 uM TmoIMR0111 0.75 0.15 uM 0.75 5 uM TmoIMR0107 0.75 0.15 uM 0.75 DNA 10.00 - 10.00

Cycling  Step # Temp °C Time Note 1 50 2 min - 2 95 10 min - 3 95 15 sec - 4 60 1 min repeat steps 3-4 for 40 cycles

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.