Stock Number: 002596 Strain Name: B6;129P2-Nos2tm1Lau/J

Allele: Nos2tm1Lau Protocol Name: Nos2tm1Lau Method: Standard PCR Version: 2.1 Created: 17-July -2008 Updated: 18-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 = 275 bp Heterozygote = 275 bp and 108 bp Wild type = 108 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3% gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1216 - ACA TGC AGA ATG AGT ACC GG - Common oIMR1217 - TCA ACA TCT CCT GGT GGA AC - Wild type oIMR1218 - AAT ATG CGA AGT GGA CCT CG - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.67 - 2.67 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 oIMR1216 0.80 1.33 uM 0.80 20 uM oIMR1217 0.05 0.08 uM 0.05 20 uM oIMR1218 1.50 2.50 uM 1.50 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 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 59 30 sec - 4 72 30 sec 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 002596 B6;129P2-Nos2tm1Lau/J 002609 B6.129P2-Nos2tm1Lau/J 004684 B6(129P2) Nos2tm1Lau-chtl/GrsrJ 007072 CByJ.129P2(B6)-Nos2tm1Lau/J 009126 B6.Cg-Nos2tm1Lau Tg(Thy1-APPSwDutIowa)BWevn/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.