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The quality of DNA is the single most important factor in obtaining high quality sequence data.

To ensure proper concentration, please check your samples by electrophoresis prior to shipping. There should be a single clear band of the appropriate size when loading 1 µl of the sample on a 1% agarose gel with EtBr. Smearing or multi-bands can be causal factors in sequencing failures. Using UV absorbance to quantify DNA may provide inaccurate measurements of target DNA concentration.

DNA should be dissolved in Nuclease-Free distilled water. Nuclease-Free super low EDTA TE buffer (10mM Tris-HCI, pH8.0, 0.01mM EDTA) can be used but with caution. If the TE buffer with EDTA (concentration of 0.1-1Mm) is used for dissolving DNA, EDTA take up free magnesium ions, which reduces DNA polymerase activity resulting in sequencing reaction failure.

Sequencing Sample/Primer Requirements
Sample Type/Format DNA size Concentration Volume
Plasmid 4 ~ 8kb 80 ~ 150 ng/µl 10 µl per reaction
8 ~ 15kb 150 ~ 200 ng/µl
PCR Product Less than 300bp 10 ~ 20 ng/µl
300bp ~ 700bp 20 ~ 30 ng/µl
Over 700bp 30 ~ 50 ng/µl
BAC 40 ~ 200kb 500 ~ 1000 ng/µl 15 µl per reaction
gDNA - 30 ~ 50 ng/µl 15 µl per sample
Premix Plasmid - 100 ng/µl 5 µl sample + 5 µl primer
Purified PCR Product - 50 ng/µl
Primer - 5 pmol/µl

Primer Primer size Concentration Volume
Primer for regular sequencing 18 ~ 27 mer
(Tm 55℃ - 60℃)
2 ~ 5 pmol/µl 10 µl per reaction
Primer for BAC sequencing 100 pmol/µl

Samples and primers can be submitted in 1.5 ml tubes, strip tubes or 96-well PCR plates.

A temperature control is not necessary in the below cases. Samples are stable for a few days at room temperature.
    -    DNA samples in water or TE
    -    Bacterial cells in agar-stab or agar plate culture