For most templates, any labeling method will work well. 32 P is recommended for situations where maximum sensitivity is required. Recommendations for the optimal labeling method for various templates are outlined in Table 1. Gives maximum sensitivity, which is required for small amounts of template. Amounts of template are usually adequate for use with any labeling method.
Internal primer is recommended to decrease sequence artifacts due to secondary products.
Quantitate your template and primer before sequencing, and use the recommended amounts of template and primer for the chosen labeling method. The molar amounts of template and primer recommended in our protocols have been empirically determined to provide the best results. Calculate the T m for your primer and use the conditions that result in optimal labeling. The optimal annealing temperature will, of course, be near the T m of the primer. Use radiolabeled products with the recommended specific activities.