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 on an agarose gel before submitting the sample.

Purification ? Use of QIAGEN Qiawell and Qiaprep kits are recommended (for dsDNA and ssDNA). The template should have an OD260/OD280 near 1.8. Make sure there is NO RNA in the prep.

Resuspension ? Use sterile deionized water (molecular biology grade). EDTA should NOT be present.

Template Amount ? The quantity of template to be used in the sequencing reaction is listed below. NOTE: too much template will result in a poor sequence. We will not be responsible for a poor sequence due to too much template!

• For dsDNA and purified PCR products, use 50 fmol/reaction
• For dsDNA plasmids, use 50 to 100 fmol/reaction
• For ssDNA, use 25 fmol
• The volume of DNA template to be used in the sequencing reaction is 10.0 ml.
  (Note: the total volume of the reaction is 20 ml)

Use the table attached to the Submission Sheet to estimate the ng needed for 50 to 100 fmol.

Template Pre-Heat Treatment ? For plasmid DNA templates, heating the template to 96° C for 1 min. will improve both signal strength and current stability. A plasmid template should be diluted in water (MB grade) and submitted separately from the primer. We will heat treat before the addition of the other reaction components.

Primer Consideration: The standard conditions for thermal cycling in the CEQ8000 DTCS protocol work well with the -47 primer used for the pUC18 control template. Other primers, such as SP6, T3, T7 and -21, are shorter and may have a less than optimal Tm for the standard method. Primers that do not meet the criteria listed below may require change in our standard thermal cycling program.

Optimal Primer Design -

• 20 ? 24 bases
• 50% GC content
• Tm optimum ³ 60° C
• No dimerization
• 5 mM stock concentration

Primer Amount - 40 to 50:1 Primer:Template ratio for dsDNA

Custom Primer (5 mM stock) = 2.0 ml /reaction