Plasmid DNA Preparation

The quality of template DNA is extremely important to obtain good sequence data. The following methodologies are recommended for preparing plasmid DNA for automated sequencing: Cesium chloride (CsCl) banding, Qiagen plasmid kit, Wizard plus plasmid kit, and other commercial kits available. Magnesium ions are essential for DNA polymerase activity. Template DNA and primer should be resuspended in water or a buffer containing no more than 0.1 mM EDTA. Introduction of large amounts of EDTA in template DNA or primer will result in weak signals or short reads. The optimal concentration of the template should be at 0.25 μg/μl.

PCR Product Preparation

The purity of the PCR product is very crucial for obtaining good sequence data. Any PCR primers and/or dNTPs remaining in the PCR product will adversely affect the quality of the sequence data. If the PCR product has a unique band, it can be purified by size exclusion method, like PCR purification kit from Wizard SV gel and PCR clean-up system or Qiagen and Pharmacia. If the PCR product has more than one band, the PCR product should be run on the agarose gel in order to isolate the desired band needed for sequencing. The band can be purified by Gel Extraction kit from Wizard (Promega) or Qiagen. The purified PCR products can then be quantitated either on the gel or spectrophotometer.

Label all samples with a unique template identifier for each template, the date and your initials.