Our Device:

Sequencing is performed by ABI PRISM® 3100-Avant Genetic Analyzer. Only reagents ordered from the manufacturer (ABI) -directly- are used, and samples are always loaded according to the default parameters. Sequence detection is based on capillary electrophoresis. Samples are labeled by a PCR reaction with fluorescently-labeled diedoxi-NTPs. As a result of chain termination different size and labeled fragments are formed, that are separated by capillary electrophoresis. Labeled fragments coming through the detection window are induced by laser, and the fluorescent signal is captured by a CCD camera.

Sequencing protocol:

Sequencing of PCR products, plasmid and gDNA samples are available. We are able to sequence till 600-700 base length, data on sequences are reliable for 600 bases. For sequencing of samples with enclosed quality control data: agarose gel photo, OD ratio >1.7, concentration warranty is provided.

After every polymer replacement the proper operation of the device is checked by running an original control sample (310/377/3100/3100-Avant™/3130/3130xl Genetic Analyzer Sequencing Standards, BigDye® Terminator v3.1). Having successful test runs warranty cannot be enforced in case of customer’s sample failure and each failed sample will be charged to the costumer. We are ready to consult you about the causes of defective samples.

Accepted sample types:

1. After sequencing reaction purified, dried product.

2. Sequencing reaction included, DNA template and primer are required in the following volumes:

Fragment analysis (microsatellite analysis, genotyping):

By D filter calibration we are ready to detect the following dyes: 6FAM, HEX, NED, ROX. 5ul PDC product is required for the runs, which contain the labeled fragments, and also 1 ul/sample for size standard is needed.

Sample preparation for sequencing:

In case of PCR products it is important to use only one specific product as the template. If there are more products the product with the appropriate size has to be cleaned from the gel.

In case of plasmids you should be sure that your colony is made of 1 clone.

Be careful not to leave any alcohol in your sample!

During PCR sequencing the samples has to be labeled by ddNTP, and only one of the primers has to be used. The labeled product must be purified to remove non-built in ddNTPs, and subsequently has to be lyophilized.

I. Sequencing:

The results of each run are saved in 2 different files types. ".seq" contains the pure sequence, and may be opened with a .txt reader. ".ab1" contains the electropherogram, and may be opened with the following "free of charge" programs:

• EditView software, Applied Biosystems (Macintosh OS 9.x, 1.5 Mbytes).

• FinchTV software (Macintosh OS X, Windows, Linux).

• 4Peaks (Macintosh OS X).

• Chromas software (Windows/DOS).

II. Fragment analysis:

The ".fsa" contains everything per sample, which may be opened with Genotyping Software, distributed by Applied Biosystems.

/Back to Functional analyses.