Location

Ready-to-run sequencing

Service description

Ready-to-Run sample batches of 96 require processing time of about 1-2 working days if samples and Sample submission form delivered before 10:00 in the morning. Individual samples (i.e. any other than full series of 96) may take longer. Expected read length (QV>20) for high quality DNA template/primer is approximately ~700 bp.
Customer provides

  •     Ready made sequencing reactions (10µl) preferably in a 96-well plate format.
  •     Completely filled sequencing submission form as e-mail attachment sent to fimm-seqlab(ät)helsinki.fi.

Ready-to-Run sequencing includes

  •     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.

Ready-to-Run Protocol

Prepare template

Poor template quality is the most common cause of sequencing problems. 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 elctrophoresis.
  •     Purify sequencing template (e.g. ExoI-SAP, EXO-SAPiT, RESIN).

See Prepare Templates for more information.

Sequencing reaction setup

  •     Prepare a sequencing master mix (see table below).
  •     Deliver 8.0 µl of master mix to each well.
  •     Add 2.0 µl of purified template to each well.
  •     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: Fill plates by columns: A1, B1, C1..>> see Plate filling help.

BigDye dilutions for different templates

Template length[bp]   PCR < 200 bp PCR 200-500 bp PRC 500-800 bp >800 bp(plasmid)
Dilution ratio [c] 1:50 1:32 1:16 1:12
Milli-Q water   5,30µl 5,20µl 5,10µl 5,00µl
BigDye Dilution buffer v3.1 5x 1,90µl 1,90µl 1,75µl 1,65µl
Bigdye RR mix v3.1   0,15µl 0,25µl 0,50µl 0,7µl
Your sequencing primer 5µm 0,65µl 0,65µl 0,65µl 0,65µl
Template (Purified)   2,00µl 2,0µl 2,00µl 2,00µl
Total rxn volume   10µl 10µl 10µl 10µl
NOTE: Dilutions have only been tested for BigDye v3.1 chemistry with original Dilution buffer from Applied Biosystems.
  •     Seal the plate properly with PCR tape.
  •     Spin samples down with centrifuge - SeqLab plate centrifuge is at your service.
  •     Proceed to thermocycling

Thermocycling

Place your samples in thermocycler and run following cycle sequencing protocol. SeqLab recommends the modified protocol to gain stronger signals.

  • Original BigDye 3.1 protocol (Applied Biosystems)
96°C for 1 min Initial denaturation
24 cycles of
  •     96°C for 10 s
  •     50°C* for 5 s
  •     60°C for 4 min
Use rapid ramp (1°C/s)
  •     Denaturation
  •     Primer annealing (50 - 60°C)
  •     Extension
10°C forever Hold until delivered to SeqLab
  • Modified BigDye 3.1 protocol (used for Full Service sequencing)
96°C for 1 min in  Initial denaturation
29 cycles of
  •     96°C for 10 s
  •     50°C* for 10 s
  •     60°C for 4 min
Use rapid ramp (1°C/s)
  •     Denaturation
  •     Primer annealing (50 - 60°C)
  •     Extension
10°C forever Hold until delivered to SeqLab
NOTE: You can modify primer annealing temperature* according to your primer (max. 60°C)
NOTE: If the samples are evaporated you can try increase volume to 10 µl with Milli-Q water.
Last updated: 25.08.2016 - 13:29