Instructions

General instructions

DNA sequencing services are located at the Biocenter Oulu facilities, Medical Campus, Aapistie 5A 90220 Oulu, Rm 448B.

Please, read the following instructions carefully, since samples without adequate information will not be processed.

Samples are placed in the cold room 442B and must be accompanied with a fully filled sequencing form (place in a box near room 448B). Please write your full name and the full name of your research group leader.

Samples will be run in the order of arrival and should be brought in before 3 pm to be run during the next working day. . Results can be obtained from server within three (3) working days. Due to the large number of researchers we have no capacity to perform sequencing reactions for individual annealing temperatures. We provide three annealing temperatures: 50°C, 55°C, and 60°C. Please take this into consideration when designing your primers.

Sample list should be provided as an excel file and sent electronically to lbkseq (at) oulu.fi. Excel file name has to include owner’s name and date. This file name should also be indicated in the sequencing form.

There is no limit for the size of the sample name, but they should NOT include Scandinavian letters, spaces or any of the following:

\ /: * ? " ‘ , < > |

Sample names should start with your initials (for example MM_1_DN13_COL1A2_e5F).

NOTE: To avoid mistakes in the analysis, please do not use the whole sample name when marking individual tubes. Your initials and running number in tubes relative to your excel file is sufficient and more easily readable.

Full service sequencing

For full service sequencing samples need to contain good quality DNA template and primer in a total volume of 6 ul (microlitres). We will perform the sequencing reactions using BigDyeTerminator v1.1 cycle sequencing kit (Applied Biosystems) and ethanol precipitation purification. Samples are run on Applied Biosystems 3500xL Genetic Analyzer.

Guidelines for template concentrations:

  • In the table below are recommendations by Applied Biosystems for template amounts to be used for PCR-products of different length.
  • In general, higher DNA quantities give higher signal intensities, but higher DNA quantities may also give shorter read lengths and top-heavy data.
  • The used amount of PCR product in sequencing depends on the length and purity of the PCR product.
  • Given amounts are guidelines and the results are affected also by your template sequence and the choice of primer.
    • Obtained signal intensities may differ for two different primers that are used for sequencing the same template.
    • For PCR products between 180-1000 bp from human genomic DNA, we generally use 10-20 ng for sequencing with good results.

PCR-product

Quantity (Applied Biosystems)

100-200 bp

1-3 ng

200-500 bp

3-10 ng

500-1000 bp

5-20 ng

1000-2000 bp

10-40 ng

>2000 bp

20-50 ng

  • Plasmid samples should have a final DNA amount of 150-300 ng (large plasmids might need 500-750 ng), Cosmid or BAC 0.5-1 ug.
  • Samples should arrive with the primer added.
  • Primer concentration is usually 5 pmol per reaction.

Ready-to-run sequencing

Ready-to-run sequencing is an option for those who prefer to do sequencing reactions and purifications themselves.

  • Sequencing reactions done with BigDyeTerminator v1.1 cycle sequencing kit (Applied Biosystems)
  • Ready-to-run samples are brought in dried.

Template purification i.e. removal of PCR reaction “residuals” (primers, dNTPs, salt, non-specific PCR products) is crucial for good quality sequence. Depending on the quality of the PCR product methods that can be used are:

  • ExoI/SAP treatment
  • Commercial column purification
  • Gel purification
  • Purification with magnetic beads (Agencourt Ampure or similar)

Fragment analysis

  • Samples for microsatellite analysis should be ready for run and contain a correct size standard in addition to labelled PCR-products in 10 ul (microlitres) of Hi-Di Formamide.
  • Matrix Standard sets DS-33 (6-FAM, VIC, NED, PET, LIZ), and DS-02 (dR110, dRGG, dTAMRA, dROX, LIZ) are available.

Forms

Obtaining results

Oulu University researchers (UNIV-users) will be able to obtain their data from:

\\lipasto\yo-yhteiset$\Projektit\BCO_ABI\ABIDATA

On the server the samples are organized in folders by year and month, then according to the electrophoresis date/time and initials of the researcher. Individual sequences will have the file name sent by the researcher.

For example:

We will e-mail the data only for non-UNIV or non-Oulu University users.

Sequencing prices

 

Academic groups

Non-academic groups

Full service sequencing

6,40 €

8,90 €

Ready-to-run sequencing

3,60 €

8,30 €

Fragment analysis

3,60 € *

8,30 € *

 

* LIZ size standard €0,80/sample

 

Prices are indicated as VAT 0%.

For additional information regarding the prices contact Katri Pylkäs (katri.pylkas (at) oulu.fi; tel. +358-(8) 315 3650).

Troubleshooting

Why my sequences did not work?

Please, consult the following link for possible solutions: Sequencing troubleshooting.

Last updated: 29.11.2018