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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 ul 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 Sample Requirements Remarks
Plasmid - 80-200 ng/µl
- 10 µl for initial reaction, 3 µl for
   each of additional reaction
DNA size 4-8kb : 80-120 ng/µl
DNA size 10-15kb : 150 - 200 ng/µl
PCR Product - 5-60 ng/µl
- 10 µl for initial reaction, 3 µl for each of
   additional reaction
Size Below 200bp : 5-10 ng/µl
Size 201bp-500bp : 20 ng/µl
Size Over 500bp: 25 ng/µl
Size Over 1000bp: 30-60 ng/µl
BAC - 500 ng/µl - 1 ug/µl
- 15 µl for initial reaction, 6 µl for
   each of additional reaction
Size 40-200 kb
16S rDNA sequencing - Agar plates or glycerol stock
- genomic DNA: 30-50 ng/µl
- 15 µl of genomic DNA, 200 µl of glycerol stock
N/A
Primer Walking - 8 µl (for an insert size of up to 4kb) Single Strand Sequencing : 1µl kb insert
If insert size is longer than 4kb,
clone is required in an agar stab
culture status.
Primer - 2-5 pmol/µl
(10pmol/µl for BAC/Cosmid samples)
- 10 µl for initial reaction, 2 µl for
   each of additional reaction
18-27 mer, Tm 55°C - 60°C
Universal Primers are available at
free of charge
Premix Plasmid 100 ng/µl
Purified PCR Product 50 ng/µl
Primer 5 pmol/µl
Premixed Total Volume 5 µl Template + 5 µl Primer

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

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