Custom DNA Sequencing:Sample Preparetion | Hokkaido System Science Co.,Ltd.

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Hokkaido System Science Co., Ltd. (HSS) delivers sequence data with very short turnaround and at competitive prices. The sequencing is based on high quality oligo-synthesis technique.
HSS strives to work as each customer's own Sequencing Dept.

Sample Preparation

Concentration Measurement

Agarose Gel Electrophoresis

Shipping volume

Please make sure to perform an agarose gel electrophoresis to confirm sample conditions and concentration before sending.
Low concentration or degradation of samples may cause the analysis difficulties and orders may no be accepted. Thank you for your understanding.

For your information, please refer to our recommended protocol as below.

Sample preparation

Please use commercially available purification kit for sample extraction and purification so that the analysis can be successfully performed. Especially for PCR products, the remaining of primers or dNTP after PCR reaction may affect the analysis result adversely.

Our recommended purification kit: Labo Pass™ Products series

Please be minded that when samples are solved with EDTA including buffers such as TE, EDTA may inhibit the cycle sequencing reaction.
It is recommended to use sterile deionized water when preparing the sample and the primer.

Concentration Measurement

To measure the sequencing sample concentration, our recommended method is Agarose gel electrophoresis.
Concentration measured by spectrophotometer may be not accurate if any remaining other than DNA is included. (Reading may differ by, as much as 10 folds.)

Therefore, please measure the sequencing sample concentration by agarose gel electrophoresis and compare the brightness with molecular weight markers.

If molecular weight markers are not available, please take an electrophoresis using 1 ?l of apply as a guide. The image to accompany the sample should show the band clearly.

Please provide the original electrophoresis image, not copied one, with the information such as apply volume and concentration of the sample and marker.

Agarose gel electrophoresis

For plasmid sample

1.0% agarose, TAE buffer, plasmid marker prepared to be 200ng/µL.
Apply volume of the marker and samples is 1.0µL each.

M 200ng 1µL	Sample1 1µL	Sample2 1µL	Sample3 1µL
				M = plasmid marker

Based on the above images, concentration for each sample is estimated as below;

  Sample 1: approx. 150ng/lane i150ng/1µLj
  Sample 2: approx. 400ng/lane i400ng/1µLj
  Sample 3: approx. 400ng or more/lane * This seems to be saturated, so another

electrophoresis with diluted sample would be recommended.

If RNA contamination is found in the agarose gel electrophoresis result, please remove RNA by RNase treatment or any other way.
Excess RNA contamination may inhabit sequencing reaction.

	<Electrophoresis result of excess RNA contaminated sample>
	<-RNA

For PCR product

2.0% agarose gel, TAE buffer, marker ("100bp DNA Ladder" from Takara Bio)
Apply volume is 4.0ƒÊL for the marker and 1.0µL for each sample.

* DNA volume of each band on this marker is readily prepared. HSS prepares each band to be
equivalent to approx. 20ng when 4µL applied.

M	Sample1	Sample2
4µL	1µL	1µL
			M = 100bp DNA Ladder

Based on the above images, concentration for each sample is estimated as below;
Sample 1: 15ng/lane(15ng/µL)
Sample 2: 60ng or more/lane

It is difficult to obtain good data from a sample which shows plural bands or primers remaining in its agarose gel electrophoresis result.
In such case, please try to excise the sample from gel or remove primers once again.

<-Primer

Shipping volume

Required sample volume for one sequencing run depends on the sample size.
Please refer to the below table for your sample preparation.

Sample volume may be reduced due to evaporation and other reasons during transport.
Please send enough volume, more than 10µL, even though the high concentration.

* For the sample with less concentration than the below, please ask to us and make the
shipping volume more.

For plasmid sample:

Sample size	Concentration required
Between 2 and 4 kbp	0.2ug/µL=200ng/µL
6kbp	0.3ug/µL=300ng/µL
8kbp	0.4ug/µL=400ng/µL
10kbp	0.5ug/µL=500ng/µL

For PCR product:

Sample size	Concentration required
Between 100 and 500 bp	10ng/µL
Between 500 and 1000 bp	Between 10 and 20ng/µL
Between 1000 and 2000 bp	Between 20 and 40ng/µL
More than 2000bp	Between 40 and 100ng/µL

Primer:

Please send 10µL or more of approximately10µM primer per reaction.