In order to obtain good sequence data the quality of the template DNA is extremely important. The following methodologies are recommended for preparing plasmid DNA for automated sequencing: Cesium chloride (CsCl) banding, Retrogen plasmid kit, Qiagen plasmid kit, Wizard plus plasmid kit, along with other commercial kits. Magnesium ions are essential for DNA polymerase activity. Template DNA and primer should be re-suspended in water or a buffer containing no more than 0.1 mM EDTA. High concentrations of EDTA in template DNA or primer will result in weak signals or short reads. The purified plasmid DNA can then be quantified either by agarose electrophoresis or by spectrophotometer. The optimal concentration of the purified plasmid template is 100 ng/μl.
PCR Product Preparation
The purity of the PCR product is crucial to 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 the size exclusion method, using a PCR purification kit from Retrogen, Qiagen or 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 the Gel Extraction kit from Retrogen or Qiagen. The purified PCR products can then be quantified either by agarose electrophoresis or by spectrophotometer. The optimal concentration of the PCR template is 20 ng/μl.
QC Your Templates
It is necessary to measure the concentration of your template accurately, either using an agarose gel or by spectrophotometer. The primary factor contributing to poor sequencing results is inaccurate concentration. So, please verify the concentration of your template accurately.
Recommended DNA Template and Primer Quantities for Each Reaction
DNA and Primer Separated
DNA
Concentration
Amount
2x Amount
PCR product
DNA 5-20ng/μl
50ng
100ng
Single stranded plasmid
plasmid 50-100ng/μl
150ng
300ng
Double stranded plasmid
plasmid 50-200ng/μl
300ng
600ng
BAC
100-200ng/μl
2000ng
4000ng
Primer
10pmol/μl
5μl
10μl
We recommend 2x amount of DNA in case we need to repeat the reactions DNA and Primer Combined
DNA
Amount
Primer
Total Vol.
2X DNA
2X Primer
2X Total Vol.
PCR product DNA
50ng
10pmoles
6μl
100ng
20pmoles
12μl
Single stranded plasmid
150ng
10pmoles
6μl
300ng
20pmoles
12μl
Double stranded plasmid
300ng
10pmoles
6μl
600ng
20pmoles
12μl
BAC
2000ng
10pmoles
6μl
4000ng
20pmoles
12μl
We recommend 2x amount of DNA in case we need to repeat the reactions.
Example 1: If your PCR template and primer are at the concentration of 20ng/μl and 10pmol/μl, respectively, you need to add 5μl of PCR template and 2μl of primer to the 1.5mL epi tube, and add 5μl of H2O to bring the total volume of 12μl.
Example2: If your Plasmid template and primer are at the concentration of 200ng/μl and 10pmol/μl, respectively, you need to add 3μl of Plasmid template and 2μl of primer to the 1.5mL epi tube, and add 7μl of H2O to bring the total volume of 12μl.
Single Tube Sequencing
Smaller orders should use 1.5mL Eppendorf tubes. Screw cap tubes are more time consuming, so pop caps should be used. Each tube must be clearly labeled to match with the order forms.
96 Well Plate sequencing (25 and More Samples Per Order)
Please submit your DNA and primers at the concentration according to the table above. The concentration of DNA and primers should be normalized and arrayed arranged vertically (A1-H1, A2-H2, etc.) on the 96 well plate. The plate should be sealed using strip-cap lids to prevent leakage and shipped overnight. Tube strips, while convenient, are hard to label and track, so for large orders (24+ samples) use 96 well plates. We recommend using strip-cap lids to seal plates as they prevent leakage and cross contamination. This is especially important for plates shipped overnight.
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