GENEWIZ, Inc. - DNA Sequencing Sample Submission Guidelines

To prepare samples for DNA sequencing, please follow these easy steps:

For orders with <48 samples, please use 8-strip PCR tubes to streamline preparation and processing. Label your tubes on the side with your initials and sample number. For orders with ≥48 samples, you can receive a discount by using a 96-well PCR plate and arranging the samples vertically (A1 to H1). See the Tubes and Plates Tab for more details.

Dilute your sequencing primer to 5 µM (pmol/µl) using water. You will need 5 µl for each sequencing reaction. If you want to use a GENEWIZ Universal Primer, we will add it for you at no charge. Remember that only one primer is used in a sequencing reaction. See the Technical Notes Tab for Tips on designing primers for sequencing.

For the amount of template needed in each of our DNA Sequencing Services (Pre-Mixed, Pre-Defined, and Custom), please refer to the tables below. Prepare template in 10 µl for each sequencing reaction. Please make dilutions in water or Tris. For best results, do not use Tris-EDTA (TE) because EDTA will inhibit the sequencing reaction.

I. Pre-Mixed

In the same tube, mix template (10 µl) and your primer (5 µl) according to the table below. To use a GENEWIZ Universal Primer, simply submit template at the requested concentration in 10 µl. See the Technical Notes Tab for Tips on how to purify PCR products.

DNA Type	DNA Length (include vector)	Template Concentration in 10 µl	Template Total Mass	Your Primer Total Picomoles	Premixed Volume* (Template + Your Primer)
Plasmids	<6 kb	~50 ng / µl	~500 ng	25 pmol	15 µl
	6 - 10 kb	~80 ng / µl	~800 ng
	> 10 kb	~100 ng / µl	~1000 ng
Purified PCR Products	<500 bp	~1 ng / µl	~10 ng	25 pmol	15 µl
	500 - 1000 bp	~2 ng / µl	~20 ng
	1000 - 2000 bp	~4 ng / µl	~40 ng
	2000 - 4000 bp	~6 ng / µl	~60 ng
	>4000 bp	Treat as plasmid	Treat as plasmid

*If you use a GENEWIZ Universal Primer, submit the required amount of template in 10 µl.

II. Pre-Defined

In separate tubes, provide template (10 µl) and your primer (5 µl) according to the table below. See the Technical Notes Tab for Tips on how to purify PCR products.

DNA Type	DNA Length (include vector)	Template Concentration in 10 µl	Template Total Mass	Template Volume Per Reaction	Your Primer Concentration µM (pmol/µl)	Your Primer Volume Per Reaction
Plasmids	<6 kb	~50 ng / µl	~500 ng	10 µl	5 µM	5 µl
	6 - 10 kb	~80 ng / µl	~800 ng
	>10 kb	~100 ng / µl	~1000 ng
Purified PCR Products	<500 bp	~1 ng / µl	~10 ng	10 µl	5 µM	5 µl
	500 - 1000 bp	~2 ng / µl	~20 ng
	1000 - 2000 bp	~4 ng / µl	~40 ng
	2000 - 4000 bp	~6 ng / µl	~60 ng
	>4000 bp	Treat as plasmid	Treat as plasmid

III. Custom

With this service, GENEWIZ will determine template concentration, optimize for sequencing, and mix with primer. Please provide template concentration if available.

Choose this option to sequence plasmids, PCR products (purified and unpurified), bacteria, phage with circular genome, and BAC DNA. For details on sequencing bacteria, phage with circular genome, and BAC DNA, please refer to the Bacteria and Phage Tab.

DNA Type	DNA Length (include vector)	Template Concentration	Template Total Mass (recommended)	Template Volume Per Reaction	Your Primer Concentration µM (pmol/µl)	Your Primer Volume Per Reaction
Plasmids	Any size	Unknown	At least 500 ng	10 µl	5 µM	5 µl
PCR Products*	Any size	Unknown	At least 40 ng	10 µl	5 µM	5 µl

*If you are sending unpurified PCR products, please send the same amount of DNA that is required for purified PCR products.

Choose the Custom service type when sequencing from a bacterial colony, glycerol stock, phage with circular genome (supernatant or plaque), or BAC DNA. For these services, please add one additional day to the turnaround time.

For each reaction, provide 5 µl of your primer at 5 pmol/µl (5 µM) in a labeled 1.5 or 0.5 ml tube.

Bacterial Colonies

Please submit bacterial clones on an agar plate wrapped in parafilm.

Labeling - Circle and label colonies you need sequenced. If you are submitting more than one plate, label each with the corresponding order tracking number. When submitting multiple plates for the same tracking number, please make sure that the sample name on the submission form clearly corresponds to the labeled plates (e.g. PlateA-Colony1).
Shipping - Send colonies on an agar plate using Standard Overnight at room temperature in a box with padding. You can also use your GENEWIZ Drop Box, please contact us for locations and pickup times. Caution: We do not receive samples on Saturdays. Please contact GENEWIZ Technical Support at 1-877-436-3949 option 2 before sending samples on a Friday.

Tip: The best way to prevent cross-contamination is by growing colonies in a grid pattern on an agar plate. This is also helpful when clones need to be tracked for use in downstream applications, such as plasmid preparation. Simply draw a grid on the back of the plate with alcohol-resistant, indelible ink.

We also sequence colonies that are randomly distributed on an agar plate. Please let us know if positive clones will be used in downstream applications, like plasmid preparation, after they have been sequenced. You can do this by writing "May need DNA prep service after sequencing- please label colonies" in the "Comments" section of the DNA sequencing order form.

Glycerol Stock Clones

Please submit 10 - 50 µl of glycerol stock per reaction.

Preparation - Prepare bacterial cultures with 8-30% glycerol in 1.5 ml or 2 ml eppendorf tubes. For sequencing many samples, use 96-well PCR or V-bottom plates.
Shipping - Glycerol stock samples must be sent on dry ice. Please do not send glycerol stock samples on Fridays because we do not receive samples on Saturdays. Please do not use your GENEWIZ Drop Box.

Phage with Circular Genome

Please submit 50 µl of phage supernatant in 8-strip PCR tubes or 1.5 ml eppendorf tubes, or provide phage plaques in a petri dish.

Titer - Please indicate the titer in the "Comments" section of the order form.
Large Projects - Please contact us to set up a pilot study. This usually consists of 3-4 dilutions to determine the optimal sequencing conditions.

BAC DNA

Be aware that sequencing BAC DNA is challenging, so we request concentrated stocks of DNA and primer.

BAC DNA - For each sequencing reaction, provide 2.5 µg or more in 15 µl. The BAC DNA concentration should be at least 150 ng/µl.
Primer - Please provide 5 µl per reaction at 10 pmol/µl (10 µM) in a labeled 1.5 or 0.5 ml tube.
Large Projects - Please contact us to set up a pilot study.

Note: Sequencing BAC DNA requires an additional day of turnaround time.

Tubes for <48 Samples

If you are submitting <48 samples, please use 8-strip, 0.2 ml PCR tubes and caps. You can cut off empty tubes and use them next time.

GENEWIZ's Online Ordering System will create tube labels using your initials and the sample number. To make it easier for you, label your tubes using these labels (see Figure to the right).

Please contact us for help finding a vendor if your laboratory does not have any 8-strip, 0.2 ml PCR tubes or strip caps.

Strip Tubes DNA Sequencing Sample
Label the side of your tubes with YOUR initials and sample number. These should match your order receipt.

Plates for ≥48 Samples

If you are submitting 48 or more samples, we recommend using a 96-well, semi-skirted PCR plate. Cap the wells with 8-strip caps. These are usually ordered separately from the plates. Be sure that the caps seal tightly! The semi-skirted plate helps to prevent the plate from bending in transit, resulting in fewer loose caps. Please ship the plate with cushioning to avoid potential damage.

Please contact us for help finding a vendor if your laboratory does not have any 96-well PCR plates or matching strip caps.

Note: If you submit ≥48 Pre-Defined samples and request the use of ≥10 primers, please submit the primers in a 96-well plate arrayed to match with the appropriate template.

Arrange your samples vertically (in columns) as shown below:

96 Well Format DNA Sequencing Sample

Tips on Designing Sequencing Primers

Please make sure that your primer can adequately bind to your template. Insufficient primer binding often leads to poor quality results.

Optimal Sequencing Primer Characteristics

Approximately 18-24 bases in length
Melting temperature (Tm) between 50 - 60 degrees
GC content should be about 45 - 55%
Have a G or C at 3' end
3' end is complementary with your template

Tip: When preparing and using primers, please remember that pmol/µl = µM.

Tips on How to Purify PCR Products

PCR products should be purified by either gel extraction and eluted in water, or by enzymatic treatment. Please use gel extraction if you have more than one product from a PCR reaction (i.e. you have more than one band).

If you have a single PCR band and would like GENEWIZ to perform the PCR clean-up step, simply order "Custom" service and choose "PCR Clean-up" from the "Special Request" column. Send the same amount of DNA that is required for purified PCR products. Also include a gel image of your PCR product with the volume loaded in each well clearly labeled, and the volume and mass of the ladder.

If you would like to perform the PCR clean-up, we recommend the ExoSAP-IT kit from USB Corporation (Cat. #78200).

Tips on How to Determine the Template Concentration for DNA Sequencing

For plasmids, multiply 10 ng/µl by the length of the template in kilobases.

Optimal plasmid concentration = 10 ng/ul x kb

Example: You have a 4.5 kb plasmid (vector + insert). Multiply 10 times the plasmid size in kb. Thus, the optimal concentration for sequencing is 45 ng/µl. Since we request template in 10 µl, you would submit 450 ng plasmid.

For purified PCR products, multiply 2 ng/µl by the length of the template in kilobases.

Optimal purified PCR product concentration = 2 ng/ul x kb

Example: You have a 700 bp PCR product. Multiply 2 times the product size in kb. Thus, the optimal concentration for sequencing is 1.4 ng/µl. Since we request template in 10 µl, you would submit 14 ng purified PCR product.