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Full Service Sequencing

Service description

Sample batches of 96 samples require processing time of about 2 working days if samples and Sample submission form delivered before 10:00 in the morning. Individual samples (i.e. any other than full series or 96) may take longer. Individual samples are also charged with higher price. Expected read length (QV>20) for high quality DNA template/primer is approximately 550-600 bp.

Customer provides:

  • DNA template-primer cocktails delivered to SeqLab fridge or by mail / courier >> see Deliver samples.
  • Completely filled sequencing submission form as e-mail attachment sent to fimm-seqlab(ät)helsinki.fi.

Full Service sequencing includes:

  • BigDye v.3.1 sequencing reaction for customer's template-primer cocktails.
  • Sequencing reaction purification: dye terminator removal with Optima DTR 96-Well filter plates.
  • Electrophoresis with ABI3730xl DNA Analyzer.
  • Basecalling with Sequencing Analysis 5.2
  • Storage of chromatograms into SeqLab database.
  • Automatic e-mail notification when results are saved to database including batch specific Search Key for easy access to results.

Full Service Sequencing Protocol

Prepare template

Low template quality is the most common cause of sequencing problems. See Prepare Templates for more information. We assume that you use DNA of known concentration in PCR and purified templates in your sequencing reactions.

  • Check DNA concentration, e.g. with NanoDrop ND-1000
  • Verify success of PCR with agarose gel electrophoresis
  • Purify sequencing template (e.g. ExoI-SAP, EXO-SAPiT, filter plates)

Template - primer cocktail:

SeqLab needs 6.6 µl of purified PCR product including 8 pmol sequencing primer to perform sequencing reactions.

For each reaction pipette:

  • 5.0 µl of template (PCR or plasmid)
  • 1.6 µl of 5µM sequencing primer
  • Mix properly
  • Seal plate and spin samples down

Applied Biosystems recommends following amounts of templates for sequencing:
Template Lenght Quantity
PCR 100-200 bp 1-3 ng
PCR 200-500 bp 3-10 ng
PCR 500-1000 bp 5-20 ng
PCR 1000-2000 bp 10-40 ng
PCR >2000 bp 20-50 ng
ssDNA 20-50 ng
Plasmid DNA 150-300 ng

NOTE: for BAC/cosmid / other large templates see BigDye Terminator v3.1 Cycle Sequencing Kit protocol.

 

Last updated: 25.08.2016 - 13:28