Sanger Sequencing

Good Sequencing Result Requires High Quality DNA

DNA Preparation

Plasmid DNA

Plasmid DNA should be free from genomic DNA, RNA and other impurities.

Various standard plasmid purification methods can be used.

Commercial plasmid purification kits (column-based) usually work well.

PCR product

PCR product must be pure and of one specific sequence in order to obtain unambiguous sequencing reads.

For best results, primers, primer-dimers and dNTPs should be removed before using the PCR products as template DNA for sequencing. Different methods can be used for such clean-up:

1. Column purification*
2. Agarose gel purification
3. Enzymatic (e.g. Exo-SAP IT) purification

*Check cleanup size cut-off of column used.

Any COLOURED PCR pre-mix reagents are not recommended as the coloured dye might affect the sequencing results.

DNA QC

DNA quality and quantity can be checked by agarose gel electrophoresis with proper DNA mass ladder.

Plasmid DNA and purified PCR product concentration can be determined by UV spectrophotometer (e.g. Nanodrop) or fluorescence-based method (e.g. Picogreen or Qubit). A260/280 ratio >1.8~2 reflects pure DNA.

Primers, primer-dimers and dNTPs contribute to A260 reading, thus concentration of PCR product with such contaminants will be overestimated by UV spectrophotometer (e.g. Nanodrop) measurement.

DNA Template and Sequencing Primer Pre-mix Guidelines

DNA template and sequencing primer must be mixed (pre-mixed) before submitting for sequencing.

Prepare 15 µL of pre-mixed for submission with the following procedures:

1. Aliquot appropriate amount of template DNA*.
2. Add 1 µL of 5 µM sequencing primer..
3. Top up to 15 µL with DNase-free water.

*The guideline shows the appropriate amount of template DNA:

It is NOT recommended to use TE or other EDTA-containing buffer in the pre-mix, also AVOID adding any divalent cations, such as Mg²⁺, Ca²⁺, and Mn²⁺, etc.

*Users submitting template-primer pre-mix OR finished cycle sequencing product should optimize the template/primer ratio OR cycle sequencing reaction condition, which is specific to the template size/content and primer binding efficiency.

CPOS cannot be responsible for sequencing failure due to sub-optimal template/primer ratio OR cycle sequencing condition.

The following standard cycle sequencing programme will be applied.

Purification & Capillary Electrophoresis

Submit 20 µL of cycle sequencing reaction product. Volume can be made up with DNase-free water.

The final volume MUST BE 20 µL or samples will be rejected. Protect cycle sequencing product from light.