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Instruction

  • According to standard operation procedure of testing and instrument operation for capillary automatic DNA sequencer, Genome Sequencing Center, Testing Laboratory, Department of Medical Research and Development (MRD-SOP-SC-001).
  • The main facility : ABI 3730 DNA Analyzer (capillary automatic DNA sequencer).
  • Please provide Plasmid, PCR products or other samples to be sequenced. And adjust the concentration of the samples and volume to the following table:

    DNA type

    DNA concentration
    (ng / £gl)

    DNA volume (£gl)

    PCR DNA (<1000 bp)

    > 20

    > 10

    PCR DNA (>1000 bp)

    > 40

    > 10

    Plasmid DNA

    > 200

    > 10

  • Please fill out two forms :
    1. DNA sequencing application form (MRD-SOP-SC-001-F02) (with DNA Template electrophoresis photo, and indicate the loading volume and the concentration of Marker)
    2. Test commissioned form (MRD-POC-003-F01)
  • Provide a variety of samples, please note:
    1. Customer must provide DNA information (such as: GC, GT, or GCGT rich) and the concentration of appropriate purified DNA samples
    2. Customer must provide appropriate primer concentration (> 5 £gM), or use standing primer (M13F, M13R, T7P, SP6, T3)
  • Quality of DNA Template is very important to sequencing results, so customers must provide high quality and "clear " Template, excluding the extra protein, RNA and other impurities. Purified DNA samples OD260/280 should be between 1.7 and 1.9, OD200/260 should be around 1.1. Lower OD260/280 means that samples contain higher level of protein content, higher OD260/280 means that higher level of RNA samples and genomic DNA; higher OD200/260 means that samples
    contain genomic DNA and Salts.
  • Sequencing primer purified by PAGE is better
    1. Concentration:> 5 £gM.
    2. No mispair.
    3. Only one binding site with template.
    4. No hairpin structure, especially in the 3 '-end.
    5. 18-24 bp in length , Tm = 55 ~ 60 ¢J.
    6. 3'- end should be end in GC, the percentage of GC between 50 to 55% is ideal.
    7. Sequencing primer should avoid four or more than four repeat sequence.
  • If you deliver the samples after 3:30 pm, that will be considered as next working day cases.
  • You could get your reports within seven working days at the collection counter. (The first working day is the next day of receiving the samples).