Stock Number: 007963 Strain Name: B6.Cg-Smn1tm2Mrph/J

Allele: Smn1tm5(Smn1/SMN2)Mrph Protocol Name: Smn1tm5(Smn1/SMN2)Mrph Method: Standard PCR Version: 1.1 Created: 14-July -2008 Updated: 30-July -2010

Expected Results: Mutant = ~86 bp Heterozygote = ~86 bp and 119 bp Wild type = 119 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR8508 - TAC CCA GAT GCA GTG CTC TTG TAG - Mutant Forward oIMR8509 - CCT TAT GGC ATA GAC ACC AAC TTC T - Mutant Reverse oIMR9111 - GTC CCT GGT CGA CAA GAA CAG - Forward oIMR9112 - ACG CTC TGC TGC TGA CTT AGG - Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.76 - 2.76 10 X AB PCR BufferII 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 oIMR9111 0.60 1.00 uM 0.60 20 uM oIMR9112 0.60 1.00 uM 0.60 20 uM oIMR8508 0.60 1.00 uM 0.60 20 uM oIMR8509 0.60 1.00 uM 0.60 5 M Betaine 1.66 0.69 M 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 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 007963 B6.Cg-Smn1tm2Mrph/J 008384 B6;129-Smn1tm5(Smn1/SMN2)Mrph/J 008604 FVB.129(B6)-Smn1tm5(Smn1/SMN2)Mrph/J 008704 B6;129-Smn1tm6(SMN2)Mrph/J 008714 B6.129-Smn1tm5(Smn1/SMN2)Mrph/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.