Stock Number: 008233 Strain Name: B6.129-Nrgntm1Kph/J

Allele: Nrgntm1Kph Protocol Name: Nrgntm1Kph Method: Standard PCR Version: 1.1 Created: 11-July -2008 Updated: 03-November -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). Expected Results: Mutant = 500 bp Heterozygote = 183 bp and 500 bp Wild type = 183 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 oIMR8493 - AGA GAG GCT GGT TCT GCA AG - Wild type Forward oIMR8494 - CCA GAA CAC GCT GAG TTC AA - Wild type Reverse oIMR8495 - GAC AGT ATC GGC CTC AGG AA - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.32 - 3.32 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 oIMR8493 0.60 1.00 uM 0.60 20 uM oIMR8494 0.60 1.00 uM 0.60 20 uM oIMR8495 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.10 0.01 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 64 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 008233 B6.129-Nrgntm1Kph/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.