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Ready-to-run sequencing Service
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 Performa DTR v3 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 template for sequencing:
| Template Length | Quantity |
| PCR100-200 bp | 1-3 ng |
| PCR 200-500 bp |
3-10 ng |
| PCR 500-1000 bp |
5-20 ng |
| PCR1000-2000 bp |
10-40 ng |
| PCR >2000bp |
20-50 ng |
| ssDNA | 25-50 ng |
| Plasmid DNA | 150-300 ng |
(from BigDye Terminator v3.1 Cycle Sequencing Kit protocol)
NOTE: Fill plates by columns: A1, B1, C1..>> see Plate filling help.
BigDye dilutions for different templates
| Template length[bp] | PCR less than 200 bp | PCR 200-500 bp | PRC 500-800 bp | over 800 bp(plasmid) | |
|
Dilution ratio |
[c] | 1:50 µl | 1:32 µl | 1:16 µl | 1:12 µl |
| Milli-Q water |
5,30 µl | 5,20 µl | 5,10 µl | 5,00 µl | |
| BigDye Dilution buffer v3.1 |
5x | 1.90 µl | 1.90 µl | 1,75 µl | 1,65 µl |
| Bigdye RR mix v3.1 |
0,15 µl | 0,25 µl | 0,50 µl | 0,7 µl | |
| Your sequencing primer |
5µM | 0,65 µl | 0,65 µl | 0,65 µl | 0,65 µl |
| Template (Purified) |
2,00 µl | 2,00 µl | 2,00 µl | 2,00 µl | |
|
Total rxn volume |
10 µl | 10 µl | 10 µl | 10 µl |
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
|
Use rapid ramp (1°C/s)
|
| 10°C forever | Hold until delivered to SeqLab |
- Modified BigDye 3.1 protocol (used for Full Service sequencing)
| 96°C for 1 min | Initial denaturation |
29 cycles of
|
Use rapid ramp (1°C/s)
|
| 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.