Genotyping Smotm1Amc

(Smo-/+; SmoΔ/+; Smonull/+)

JDW 3/2012 MGI:2176255 Note: These are the targeted allele from Zhang XM, Ramalho-Santos M, McMahon AP. “Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell. 2001.

In this targeting vector, a 400 bp region (from 44 bp upstream to 358 bp downstream of the ATG start codon) was replaced by the neo cassette generating a null allele. For the wild-type Smo allele =280 bp Smo-5′: 5′-GGC CTG CGC TGC TCA ACA TGG-3′ Smo-exon 1: 5′-GCA ACT GCA GCA GCC TCC GG-3′ For the Del Smo allele, =380 bp Smo-5′: PGK-neo REV: 5′-GGC GCT ACC GGT GGA TGT GG-3′ The PCR conditions were 1 min at 94°C, 1 min at 66°C, and 1 min at 72°C for 35 cycles.

From the Reiter lab: JDW 7 (Smo F New): 5’-CAC CGG TCG CCT AAG TAG C JDW 8 (Smo R WT New) 5’-CCA GCA GTA CCA GCA GCA G JDW 9 (Smo R MT New) 5’-GCC AGA GGC CAC TTG TGT AG WT=190 MT=240 *Run WT and MT reactions separately. 2.5 ul 10X Coral Red Buffer X ul For X ul Rev 0.5 ul 10 mM dNTPs 0.25 ul Qiagen Taq X ul ddH2O 94°C, 5:00 94°C, 0:30 66°C, 0:30 72°C, 0:20 40 cycles 72°C, 1:00 16°C, forever