To prepare samples for DNA sequencing, please follow these easy steps:
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.
*If you use a GENEWIZ Universal Primer, submit the required amount of template in 10 µl.
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.
With this service, GENEWIZ will determine
template concentration, optimize for sequencing, and mix with primer. Please provide
template concentration if available.
*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.
Please submit bacterial clones on an agar plate wrapped in parafilm.
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.
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.
Be aware that sequencing BAC DNA is challenging, so we request concentrated stocks of DNA and primer.
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
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
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:
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
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/µl 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/µl 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.