Stock Number: 005439 Strain Name: B6.129S-Mecp2tm1Hzo/J

Allele: Mecp2tm1Hzo Protocol Name: Mecp2tm1Hzo Method: Standard PCR Version: 1.1 Created: 30-January -2006 Updated: 24-September-2009

Notes: This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase). Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands. The Mecp2 gene is X-LINKED. Males with the targeted allele are hemizygous mutants Expected Results: Mutant = 318 bp Heterozygote = 318 bp and 396 bp Wild type = 396 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR3912 - AAC GGG GTA GAA AGC CTG - Common oIMR3913 - TGA TGG GGT CCT CAG AGC - Wild type oIMR3914 - ATG CTC CAG ACT GCC TTG - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.42 - 3.42 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 oIMR3912 0.60 1.00 uM 0.60 20 uM oIMR3913 0.60 1.00 uM 0.60 20 uM oIMR3914 0.20 0.33 uM 0.20 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.20 0.02 U/ul 0.20 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.20 0.02 U/ul 0.20 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 62 1 min - 4 72 1 min 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 005439 B6.129S-Mecp2tm1Hzo/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.