Stock Number: 006849 Strain Name: B6.129P2-Mecp2tm2Bird/J

Allele: Mecp2tm2Bird Protocol Name: Mept2tm2bird Method: Melt Curve Analysis Version: 2.0 Created: 26-May -2009 Updated: 16-November -2009

Notes: Melting curve analysis is done using a Roche Light Cycler 480. Expected Results: Mutant Tm = 86.5°C +/- 1.0°C Wild type Tm = 84.5°C+/- 1.0°C

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7171 - AAC AGT GCC AGC TGC TCT TC - Common oIMR7172 - CTG TAT CCT TGG GTC AAG CTG - Wild type Reverse oIMR7415 - GCC AGA GGC CAC TTG TGT AG - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.80 - 2.80 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR7171 0.60 1.00 uM 0.60 20 uM oIMR7172 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 69 30 sec - 4 72 30 sec 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 2.80 - 2.80 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR7171 0.60 1.00 uM 0.60 20 uM oIMR7415 0.60 1.00 uM 0.60 DNA 2.00 0.00 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 006849 B6.129P2-Mecp2tm2Bird/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 - - Same as Reaction A