Three-week-old mice were marked by ear clipping.4 The ear clips were transferred into 1.5-l microcentrifuge tubes containing 150 l of digestion solution (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 0.1% SDS, and 1 mg/ml proteinase K). The tubes were incubated overnight at 55C on a rotating platform. Genomic DNA was precipitated by adding 50 l of 10 M ammonium acetate and 600 l of ice-cold ethanol. After centrifuging at 15,000 x g for 10 minutes, the pellets were washed with cold 70% ethanol, air dried, and dissolved in 20 l of TE buffer (5 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA). The yield was 0.5 to 2.5 g of genomic DNA per sample.
To evaluate whether genomic DNA isolated from ear clips is suitable for amplifying large DNA fragments with TaqPlus Long DNA polymerase, we genotyped the smooth muscle myosin heavy chain (SMHC) gene (Figure 1).
Two microliters of DNA were added to 16 l of PCR mixture [2 l of 10X TaqPlus Long low-salt PCR buffer (Stratagene), 2 l of dNTPs (2.0 mM each), 11.8 l H2O, and 0.2 l (1 U) of TaqPlus Long DNA polymerase (Stratagene)]. To amplify DNA fragments larger than 4 kb, 2 l of 10X TaqPlus Long PCR high-salt buffer (Stratagene) were used instead of the 10X low-salt buffer. Reactions were initiated using a hot-start protocol.
The first PCR cycle included two steps: For the first step, the tubes were incubated at 85C for 20 minutes [after 2 minutes at 85C, 2 l of a mixture of the appropriate upstream and downstream primers (to a final concentration of 0.25 M each) were added to each tube]. In the second step, the tubes were incubated at 94C for 2 minutes.
After the first cycle, the mixtures were cycled 45X at