 |
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
|
|
