SeqXcel, Inc.

DNA Sequencing

DNA samples can be submitted in four different formats.

Template and Primer Separately
Template Only
Premixed Template and Primer
Ready-to-Load Samples

Note: Before submitting samples, please review our Sample Preparation information. It describes how to prepare DNA templates and primers for use in sequencing.

Template and Primer Separately:
Submit DNA template and primer in separate tubes.

Templates
Each DNA template should be submitted in a separate microcentrifuge tube. Multiple aliquots of a single template can be pooled into one tube. If submitting templates in a 96-well plate, please attach a legend that indicates the location of each sample.

Note: Please contact SeqXcel before submitting large DNA templates for the first time. Protocol optimization may be required for these templates.

Templates	DNA Volume*	DNA conc. ( ng/µL)
PCR (< 2Kb)	≥10 µL	10 to 20 ng/µL
PCR (> 2Kb)	≥10 µL	20 to 30 ng/µL
Plasmids	≥10 µL	100 to 200 ng/µL
dsDNA (> 15 Kb)	≥10 µL	100 to 200 ng/µL
Bacterial (genomic)	≥10 µL	200 to 300 ng/µL
ssDNA	≥10 µL	50 to 100 ng/µL

* SeqXcel requests that DNA templates and primers have sufficient DNA to perform one sequencing reaction and allow us to retain one back-up sample.

These recommendations were developed to consistently produce the best possible sequencing data. We will accept DNA templates at less than the recommended amounts, but the resulting data varies greatly in quality and accuracy.

Primers
Each primer should be submitted in a separate microcentrifuge tube. Multiple aliquots of a single primer can be pooled into one tube.

The amount of primer necessary for each sequencing reaction is shown in the following table:

Template	Primer
PCR products	3.2 pmol
Plasmids	3.2 pmol
BAC's	8 to 15 pmol
Fosmids	8 to 15 pmol
ssDNA	3.2 pmol

For example, 3 to 5 µL of a 10 µM solution of primer is sufficient for one sequencing reaction.

Templates Only:
Submit DNA templates and SeqXcel will provide universal primers at no extra charge.

Check SeqXcel’s list of available primers before submitting a template only sample.

Each DNA template should be submitted in a separate microcentrifuge tube. Multiple aliquots of a single template can be pooled into one tube. If submitting templates in a 96-well plate, please attach a legend that indicates the location of each sample.

Note: Please contact SeqXcel before submitting large DNA templates for the first time. Protocol optimization may be required for these templates.

Templates	DNA Volume*	DNA conc. ( ng/µL)
PCR (< 2Kb)	≥10 µL	10 to 20 ng/µL
PCR (> 2Kb)	≥10 µL	20 to 30 ng/µL
Plasmids	≥10 µL	100 to 200 ng/µL
dsDNA (> 15 Kb)	≥10 µL	100 to 200 ng/µL
Bacterial (genomic)	≥10 µL	200 to 300 ng/µL
ssDNA	≥10 µL	50 to 100 ng/µL

* SeqXcel requests that DNA templates have sufficient DNA to perform one sequencing reaction and allow us to retain one back-up sample.

These recommendations were developed to consistently produce the best possible sequencing data. We will accept DNA templates at less than the recommended amounts, but the resulting data varies greatly in quality and accuracy.

Premixed Template and Primers:
Submit DNA template mixed with primer.

Customers can provide 12 µL of premixed DNA template and primer.

If you choose this option, please follow the procedure below to ensure the best results. Do not submit samples that are more concentrated than recommended. More is not better. Highly concentrated samples can reduce the read length of your sequencing data.

Step 1:

Aliquot the optimal amount of template into a microcentrifuge tube or plate well. The following table indicates concentration ranges that produce the best results.

Template	DNA per 12 µL
PCR Products	20 to 30 ng per 100 base pairs
Plasmids	500 ng to 1 µg
BAC's	5 to 10 µg
Fosmids	1 to 2 µg
ssDNA	500 ng to 1µg

Step 2:

Add the appropriate amount of primer, as indicated below:

Template	Primer
PCR Products	6.4 pmol
Plasmids	6.4 pmol
BAC's	15 to 30 pmol
Fosmids	15 to 30 pmol
ssDNA	6.4 pmol

Step 3:

Adjust the sample volume to 12 µL by adding sterile, deionized H2O.

Step 4:

Be sure to check the “YES” column under the “Premix” heading on your order form.

Step 5:

If submitting templates in a 96-well plate, please attach a legend that indicates the location of each sample.

Ready-to-Load Samples:
Submit samples processed using the BigDye® Terminator cycle sequencing kit and purified according to Applied Biosystems DNA sequencing protocols.

This option requires customer expertise with Applied Biosystems’ reagents and protocols in preparing samples for DNA sequencing. Samples must be completely dried in a microcentrifuge tube or 96-well plate.