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.

Last updated: 13/9/2016