Stock Number: 003262 Strain Name: STOCK Tg(Trp53A135V)L3Ber/J

Allele: Tg(Trp53A135V)L3Ber Protocol Name: Tg(Trp53A135V)L3Ber Method: Restriction Enzyme Digest Version: 2.1 Created: 12-March -2001 Updated: 24-September-2009

Expected Results: Transgene = three digested fragments (does not have 247 bp band) Internal positive control = two digested fragments (includes 247 bp band)

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

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1237 - GGA GGA GCC AGG CCT AAG AGC AA - Common oIMR1238 - ACA CCT GAT CGT TAC TCG GCT TGT C - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 4.23 - 4.23 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 oIMR1237 0.48 0.80 uM 0.48 20 uM oIMR1238 0.48 0.80 uM 0.48 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.03 0.01 U/ul 0.03 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 10 X Hph I Buffer 1.20 - 1.20 ddH2O 5.68 - 5.68 10 mg/ml BSA 0.12 - 0.12 Hph I 1.00 - 1.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 003262 STOCK Tg(Trp53A135V)L3Ber/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.