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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.
| 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 |
- 5-40 ng/µl
- 10 µl for initial reaction, 3 µl for
each of additional reaction
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Size 0.5-1 kb : 5-20 ng/µl
Size 1-2 kb : 20-40 ng/µl |
| BAC |
- 500 ng/µl - 1 ug/µl
- 15 µl for initial reaction, 6 µl for
each of additional reaction
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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
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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.
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| Primer |
- 2-5 pmol/µl
- 10 µl for initial reaction, 2 µl for
each of additional reaction |
18-27 mer, Tm 55°C - 60°C
32 universal primers are available at
free of charge |
| Premix |
| Plasmid + Primer |
- 8 µl (600 ng-1.6 µl)
+ 4 µl (8 pmol) |
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| Purified PCR product + primer |
- 8 µl (40-320 ng) + 4 µl
(8 pmol) |
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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.
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| - Sealing
of 96 Well Plates |
- Strip caps are recommended to seal 96-well
plates.
- Carefully seal the 96 well plates with strip caps (8-strips or 12-strips) or
polypropylene films
(relatively thick and malleable).
- Aluminum sealing or polyester films are not recommended.
Place samples into strip-capped well plate as shown below.
To avoid potential damage, please use out-skirted well plate.
Seal tightly to avoid sample evaporation
or contamination during transit.
Unsatisfactory results due to improper sample
preparation or shipping by customer will
be charged for re-sequencing. |
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