Stock Number: 003651 Strain Name: C57BL/6-Tg(Zp3-cre)93Knw/J

Allele: Esr2tm1Unc Protocol Name: Esr2tm1Unc Method: Melt Curve Analysis Version: 3.0 Created: 13-May -2009 Updated: 16-November -2009

Notes: Melting curve analysis is done using a Roche Light Cycler 480. Expected Results: Mutant Tm = 85.5°C +/- 1.0°C Wild type Tm = 83.5°C+/- 1.0°C

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR3145 - GTT GTG CCA GCC CTG TTA CT - Wild type oIMR3146 - TCA CAG GAC CAG ACA CCG TA - Common oIMR3147 - GCA GCC TCT GTT CCA CAT ACA C - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.20 - 2.20 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR3145 0.60 1.00 uM 0.60 20 uM oIMR3146 0.60 1.00 uM 0.60 20 uM oIMR3147 0.60 1.00 uM 0.60 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 5 min - 2 94 15 sec - 3 54 30 sec - 4 72 30 sec 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 003651 C57BL/6-Tg(Zp3-cre)93Knw/J 004745 B6.129P2-Esr2tm1Unc/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.