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Sample Preparation
How to Prepare Sequencing Samples

Valid for the European Eurofins MWG Operon sequencing facility, only.

Sample Preparation for Value Read Service in Tubes or Plates

  • It is possible to send either a stab or glycerol culture with the selected E.coli clone, the purified plasmid DNA or your unpurified or purified PCR product
  • Please use 1.5 ml tubes (no additional sealing with Para film) or 96well microtiter plates for your samples and primers
  • For samples in tubes we recommend to dissolve the DNA either in doubled distilled water or in 5 mM Tris-HCl (pH 8.0 – 9.0) without using Parafilm
  • Plates may contain a mixture of plasmids and PCR products as long as no purification is needed
  • For optimal results the concentration/amount should be normalised across the plate
  • Primers may be arrayed in a separate primer plate according to the sample layout
  • Plate position H12 should be kept free for internal quality control (optional)

Concentrations:

  • Purified plasmid DNA:
    50-100 ng/µl in a minimum volume of 15 µl
  • Purified PCR products:
    < 300 bp:            2 ng/µl in a minimum volume of 15 µl
    300-1000bp:      5 ng/µl in a minimum volume of 15 µl
    > 1000 bp:        10 ng/µl in a minimum volume of 15 µl
    The amount should be doubled for unpurified products
  • Primer Concentration
    2 pmol/µl, minimum total volume: 15 µl (corresponds to an amount of 30 pmol)

Premixed Samples in Tubes or Plates:

  • Purified plasmid DNA:
    50-100 ng/µl in a minimum volume of 15 µl plus 15pmol primer
  • Purified PCR products:
    < 300 bp:            2 ng/µl in a minimum volume of 15 µl
    300-1000bp:      5 ng/µl in a minimum volume of 15 µl
    > 1000 bp:        10 ng/µl in a minimum volume of 15 µl

Sample Preparation for Ready to Load Sequencing Service in Tubes or Plates

  • Please use 1.5 ml tubes or 96well microtiter plates
  • Please use ABI Big Dye Terminator Kit (v3.1)
  • Big Dye volume per reaction should not exceed 1 µl
  • Total reaction volume of 10 to 20 µl
  • Shipment of samples on dry ice is strongly recommended.

Sample Preparation for Sequencing Service à la Carte

  • It is possible to send either a stab or glycerol culture with the selected E. coli clone, the purified DNA, your unpurified or purified PCR product or genomic DNA
  • Please use 1,5 ml tubes for your samples and primers
  • We recommend to dissolve the DNA either in double distilled water or in 5 mM Tris-HCl (pH 8.0 – 9.0) without using Parafilm

Concentrations:

  • Purified plasmid DNA:
    50-100 ng/µl, minimum 1 µg per reaction;
    Please send double amount for samples with hairpin- or secondary structures, high GC content or other difficult regions
  • BAC-, PAC-, Cosmid-, Fosmid-DNA:
    200-1000 ng/µl, minimum 4-6 µg per reaction
  • Purified PCR products:
    < 300 bp:            2 ng/µl in a minimum volume of 15 µl
    300-1000bp:      5 ng/µl in a minimum volume of 15 µl
    > 1000 bp:        10 ng/µl in a minimum volume of 15 µl
    The amount should be doubled for unpurified products
  • Genomic DNA
    5-50 ng/µl, minimum amount is 200 ng per reaction
  • Primer concentration
    2 pmol/µl, minimum volume per reaction is 10 µl (corresponds to an amount of 30 pmol)

Sample Preparation for Walking Services

  • It is possible to send either a stab or glycerol culture with the selected E.coli clone, the purified plasmid DNA or your unpurified or purified PCR product
  • Please use 1,5 ml tubes for your samples
  • If available, please send a reference sequence in electronic form

Concentration:

  • Minimum concentration: 
    50-100 ng/µl for plasmids and 2 -10 ng/µl for PCR products
  • Minimum total amount:
    Single Strand Sequencing:
    1 µg/1 kb insert or 100 ng/1kb PCR product
    Double Strand Sequencing: 2 µg/1 kb insert or 200 ng/1kb PCR product

Sample Preparation for GLP-Compliant Sequencing Service

  • It is possible to send either a stab or glycerol culture with the selected E. coli clone, the purified plasmid DNA or your unpurified or purified PCR product
  • Please use 1,5 ml tubes for your samples
  • If available, please send a reference sequence in electronic form

Concentration:

  • Minimum concentration: 50-100 ng/µl
  • Minimum total amount for 2x coverage: 2 µg/ 1 kb insert or 300 ng/ 1kb PCR product
  • Minimum total amount for 4x coverage: 4 µg/ 1 kb insert or 600 ng/ 1kb PCR product