Sample Preparation Guidelines In order to optimize your template and primer for the Applied Biosystems BigDye® Terminator v3.1 chemistry, please consider the following: DNA Template: Template preparation is the most critical factor in obtaining good sequencing data. If you are unsure of the quality of your DNA, please check it on an agarose gel or with the Agilent 2100 Bioanalyzer before submitting the sample. Purification - QIAGEN's QIAwell and QIAprep kits are recommended for purifying plasmids and the QIAquick kit for PCR cleanup. The template should have an OD260/OD280 ratio close to 1.8. Make sure there is NO RNA in the sample. Quantification - The quantity of template to be used in the sequencing reaction is given below. Quantification can be achieved by spectrophotometric determination (see Procedures) or by commercial assay kits such as Quant-it by Molecular Probes. Alternatively, we suggest you consider using the Nanodrop 8000 instrument in 352 CBLS. The Nanodrop spectrophotometer uses only 1 µl of sample in determining both the concentration and purity of DNA and can also measure up to eight samples simultaneously. Resuspension & Dilution - Use sterile deionized water (molecular biology grade) or 10 mM Tris-HCL (pH 8.5). EDTA should NOT be present as it can inhibit the sequencing reaction. Target amounts for for dsDNA templates: 2.5 ng DNA / 100 bases per reaction for PCR products 300-500 ng DNA per reaction for plasmids Primer: The sequencing primer should be used at a concentration of 5.0 pmol per reaction. Primers can be ordered from a number of different vendors. Please follow the suggestions below in order to optimize your primer. Selecting Sequencing Primers - The choice of sequencing primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions with this kit. These decisions are particularly important when sequencing is done on real-time detection systems where signal strength is critical. Some of the recommendations given here are based on information that is general knowledge, while others are based on practical experience gained by Applied Biosystems scientists. Optimizing Primer Selection - The following recommendations are provided to help optimize primer selection: Primers should be at least 18 bases long to ensure good hybridization. Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided. Keep the G-C content in the range 30?80%. For cycle sequencing, primers with melting temperatures (Tm) above 45 °C produce better results than primers with lower Tm. For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm >45 °C. Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA. Avoid primers that have secondary structure or that can hybridize to form dimers. Several computer programs for primer selection are available. They can be useful in identifying potential secondary structure problems and determining if a secondary hybridization site exists on the target DNA. We suggest that you consider using Lasergene's Primer Select for this task (see Lasergene under the Services menu) The BigDye® Terminator v3.1 Cycle Sequencing Kit - Protocol (©2002 Applied Biosystems) also contains the following information which may help in your sample preparation: DNA Quality: Poor Template Quality Poor template quality is the most common cause of sequencing problems. The following are characteristics of poor quality templates: Noisy data or peaks under peaks No usable sequence data Weak signal Always follow recommended procedures to prepare templates. Contamination Potential contaminants include: Proteins RNA Chromosomal DNA Excess PCR primers, dNTPs, enzyme, and buffer components (from a PCR amplification used to generate the sequencing template) Residual salts Residual organic chemicals such as phenol, chloroform, and ethanol Residual detergents Determining DNA Quality The following methods can be used to examine DNA quality: Agarose gel electrophoresis. Purified DNA should run as a single band on an agarose gel. Note: Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear. Spectrophotometry. The A260/A280 ratio should be 1.7 to 1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. Agarose gels reveal the presence of contaminating DNAs and RNAs, but not proteins. Spectrophotometry can indicate the presence of protein contamination, but not DNA and RNA contamination. Agilent 2100 Bioanalyzer: Agilent's Lab-on-a-Chip technology utilizes a network of channels and wells that are etched onto a glass or polymer chip for sizing, quantification and quality control of DNA, RNA, and proteins. Only 1 µl of sample is used in each analysis with 12 samples analyzed per chip. Results are delivered within 30-40 minutes in high quality digital data and can be shown in gel-like image, electropherogram and tabular formats.