Stock Number: 010770 Strain Name: B6.129P2(Cg)-Hprttm34(Ple186-EGFP)Ems/Mmjax

Allele: Hprt1tm34(Ple186-EGFP)Ems Protocol Name: Hprt1tm34(Ple186-EGFP)Ems Method: Standard PCR Version: 3.0 Created: 22-March -2010 Updated: 22-March -2010

Notes: The Hprt1 gene is X-LINKED. Males with the targeted allele are hemizygous mutants. Expected Results: Mutant = 558 bp Heterozygote = 558 bp and 371 bp Wild type = 371 bp

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type 9662 - GGC CGG TGG AAG GAT TAT GA - Mutant Forward oIMR9434 - CCG TGA AAG GGT TTC CCA CA - Mutant Reverse oIMR9435 - AGA TGA GCT TGG TTG CTG GAG TG - Wild type Forward oIMR9436 - TGC TGG ACA TCC CTA CTA ACC CA - Wild type Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR9436 0.30 0.50 uM 0.30 25 mM MgCl2 0.96 2.00 mM 0.96 20 uM oIMR9435 0.30 0.50 uM 0.30 DNA 2.00 - 2.00 20 uM 9662 0.80 1.33 uM 0.80 10 X AB PCR BufferII 1.20 1.00 X 1.20 ddH2O 2.96 - 2.96 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 20 uM oIMR9434 0.80 1.33 uM 0.80

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 se - 3 62 1 min - 4 72 30 sec - 5 - - Go to step 2-4, 35 times 6 72 2 min - 7 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 010770 B6.129P2(Cg)-Hprttm34(Ple186-EGFP)Ems/Mmjax Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.