Stock Number: 003138 Strain Name: B6;129-Hprt1tm1Detl/J

Allele: Hprt1tm1Detl Protocol Name: Hprt1tm1Detl Method: Standard PCR Version: 1.1 Created: 15-March -2001 Updated: 24-September-2009

Notes: 10X AM Buffer: 67mM Tris.HCL pH8.8, 16.6mM NH4SO4, 2.0mM MgCl2, 0.17mg/ml BSA. Dissolve in 9 ml TE, pH to 8.8, then bring final volume to 10 ml with TE (NOT water!) Filter sterilize. Add 2-mercaptoethanol to 10 mM prior to use. This assay works well with the use of a Hotstart. Expected Results: Mutant (-/- or -/Y) = 600 bp Heterozygote = 600 bp and 98 bp Wild type (+/+ or +/Y) = 98 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.**

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0774 - CTA TAG GAC TGA AAG ACT TGC - - oIMR0775 - CAA AGA ACT TAT AGC CCC CC - -

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 6.02 - 6.02 10 X AM Buffer 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 oIMR0774 0.30 0.50 uM 0.30 20 uM oIMR0775 0.30 0.50 uM 0.30 DMSO 1.20 - 1.20 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 1.00 - 1.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 58 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 8 72 2 min - 9 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 003138 B6;129-Hprt1tm1Detl/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.