Stock Number: 008704 Strain Name: B6;129-Smn1tm6(SMN2)Mrph/J

Allele: Smn1tm6(SMN2)Mrph Protocol Name: Smn1tm6(SMN2)Mrph D Allele Method: Melt Curve Analysis Version: 1.0 Created: 05-June -2009 Updated: 18-March -2010

Expected Results: Mutant Tm = 82.5°C Wild type Tm = 85°C

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 oIMR9111 - GTC CCT GGT CGA CAA GAA CAG - Wild type Forward oIMR9112 - ACG CTC TGC TGC TGA CTT AGG - Wild type Reverse oIMR9289 - GAT GGC TTT CTG ATG CTA CAA CAC - Mutant Forward oIMR9290 - CCA ATA TTC ACA GAG GCC AAC TG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 1.24 - 1.24 2 X High Resolution Melt Master Mix (Roche) 6.00 1.00 X 6.00 25 mM MgCl2 0.96 2.00 mM 0.96 20 uM oIMR9289 0.60 1.00 uM 0.60 20 uM oIMR9290 0.60 1.00 uM 0.60 20 uM oIMR9111 0.30 0.50 uM 0.30 20 uM oIMR9112 0.30 0.50 uM 0.30 DNA 2.00 0.00 2.00

Cycling  Step # Temp °C Time Note 1 94 10 min - 2 94 30 sec - 3 62 1 min - 4 72 45 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 007246 B6;129-Smn1tm2Mrph/J 007964 FVB.Cg-Smn1tm3(SMN2/Smn1)Mrph/J 007966 B6.Cg-Smn1tm3(SMN2/Smn1)Mrph/J 008704 B6;129-Smn1tm6(SMN2)Mrph/J 009378 B6.129-Smn1tm6(SMN2)Mrph/J 009381 FVB.Cg-Smn1tm6(SMN2)Mrph/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.