Stock Number: 003890 Strain Name: B6.129P2(C)-Mecp2tm1.1Bird/J

Allele: Mecp2tm1.1Bird Protocol Name: Mecp2tm1.1Bird Method: Separated PCR Version: 2.1 Created: 09-June -2003 Updated: 24-September-2009

Notes: The Mecp2 gene is X-LINKED. Males with the targeted allele are hemizygous mutants. Expected Results: Mutant = ~ 400 bp Heterozygote = ~ 400 bp and 416 bp Wild type = 416 bp Internal positive control = 324 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel. Do not pool as both bands are approximately

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1436 - GGT AAA GAC CCA TGT GAC CC - Common oIMR1437 - TCC ACC TAG CCT GCC TGT AC - Mutant oIMR1438 - GGC TTG CCA CAT GAC AA - Wild type oIMR7338 - CTA GGC CAC AGA ATT GAA AGA TCT - Internal Positive Control Forward oIMR7339 - GTA GGT GGA AAT TCT AGC ATC ATC C - Internal Positive Control Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.56 - 2.56 10 X PCR Buffer II (ABI) 1.20 0.92 X 1.20 25 mM MgCl2 0.96 1.85 mM 0.96 2.5 mM dNTP 0.96 0.19 mM 0.96 20 uM oIMR1436 0.70 1.08 uM 0.70 20 uM oIMR1437 0.70 1.08 uM 0.70 20 uM oIMR7338 0.60 0.92 uM 0.60 20 uM oIMR7339 0.60 0.92 uM 0.60 5 mM DNA Loading Dye 1.66 0.64 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.02 U/ul 0.06 DNA 3.00 - 3.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 60 1 min - 4 72 1 min 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 2.56 - 2.56 10 X PCR Buffer II (ABI) 1.20 0.92 X 1.20 25 mM MgCl2 0.96 1.85 mM 0.96 2.5 mM dNTP 0.96 0.19 mM 0.96 20 uM oIMR1436 0.70 1.08 uM 0.70 20 uM oIMR1438 0.70 1.08 uM 0.70 20 uM oIMR7338 0.60 0.92 uM 0.60 20 uM oIMR7339 0.60 0.92 uM 0.60 5 mM DNA Loading Dye 1.66 0.64 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.02 U/ul 0.06 DNA 3.00 - 3.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 003890 B6.129P2(C)-Mecp2tm1.1Bird/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 94 3 min - 2 94 30 sec - 3 60 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold