Poor template DNA quality is the most common cause of sequencing problems affecting both sequence length and quality. Before bringing any samples to us we assume that your samples have gone through at least following quality control steps:
Quantitation of DNA samples
For genomic DNA (gDNA) samples before PCR amplifications
For plasmid DNA samples before sequencing reactions
Agarose gel electrophoresis for PCR products
Purification of sequencing template
PCR primers, excess nucleotides and salts has to be removed from the sequencing template
- e.g. ExoI-SAP purification for PCR products
- Rapid PCR purification by resin & filter plates
- Plasmid purification kits.
About DNA quantity and purity in capillary electrophoresis
The total amount of DNA in electrophoresis with ABI3730xl DNA Analyzer is a major factor affecting the quality of the results. In ABI3730xl capillary sequencing instrument samples are transferred from 96-well plate to the capillaries with an electrokinetic injection.
- Electrokinetic injection transfers negatively charged DNA (and all other negative charged) molecules to the capillaries. All DNA (labeled & unlabeled) competes in injection.
- Too high template DNA amount (PCR or plasmid etc) in sequencing reactions will compete with the fluorescently labeled sequencing reaction products in the injection thus lowering the intensity of the detected signal. Raw signal of the chromatogram may also look imbalanced due to competing molecules.
- Too low template DNA amount can lead to poor cycle sequencing reaction resulting as very low signal detection in electrophoresis.
- Other sample impurities such as excess salt or proteins will also disturb the injection.
- Some PCR buffers contain high concentration of BSA (bovine serum albumin) and had been shown to disturb the injection.