Our Sanger sequencing service can be used to sequence plasmid templates (30-100 ng/µL), purified and unpurified PCR products (10-50 ng/µL), bacterial colonies (petri dishes or 96-well agar plates), and glycerol stocks (submitted in 96-well plates) and low concentration plasmids (10 ng/µL, EU customers only).

Templates can be submitted in one of the following formats according to service type: 

• Sanger sequencing in tubes: Submission in 1.5 mL flip cap reaction tubes and affix the barcode to the tubes as indicated in our Sanger Sequencing Sample Submission Guidelines. Please do not submit in 0.5 mL, 2 mL flip cap reaction tubes, or screw cap tubes and do not wrap your tubes in parafilm.
  
  
• Sanger sequencing in plates: Submission in semi-skirted PCR 96-well plates. Submit one primer tube or one 96-well primer mirror plate per Pre-Defined sequencing plate with each well filled with the corresponding primer. As this only applies for the Sanger sequencing plate service (full-plate service with plate barcodes), invoice is always generated for a full plate; be sure to properly seal your plate, e.g. using 8-cap PCR stripes.
  
  
• Sanger sequencing Vario plate: Flexible submission in semi-skirted PCR 96-well plates. Submit up to four primer tubes per well of a pre-defined plate, reaction-based invoicing; be sure to properly seal your plate, e.g. using 8-cap PCR stripes.

 

Note: Bacterial colonies and glycerol stocks can only be submitted via our Sanger sequencing plate or Sanger sequencing Vario plate services.

Pre-Mixed service types:

• Our customers submit their template DNA and corresponding primer already “Pre-Mixed” in the same 1.5 mL flip cap reaction tube or in the same well of a 96-well plate.
• For samples submitted for Pre-Mixed service type (template DNA + primer), no reaction repeat, as GENEWIZ will only perform the sequencing without storage of the samples for repeat.

 

Pre-Defined service types:

• Our customers submit their template DNA in a 1.5 mL flip cap reaction tube or in a well of a 96-well plate and corresponding primer in a separate 1.5 mL tube.
• Alternatively, customers can also choose from different available FREE GENEWIZ Universal Primers for sequencing.
• Customers can furthermore order their customized primer to be synthesized by GENEWIZ and to be stored in the corresponding GENEWIZ facility (“Oligo Retain for Sanger”).

 

Reactions that fail GENEWIZ quality control:

For our Pre-Defined services, if the reaction does not pass our quality control (QC) in the first sequencing run, our Technical Support team initiates some test runs (straight repeats, repeats with alternative protocol, dilute repeats and/or repeat with higher amount of template DNA) to review if we can improve the sequencing results. If results can be improved, we will initiate second runs of all remaining reactions with the best working condition and/or protocol.

For orders of full plates with our Pre-Defined service, which are mainly used as high-throughput submissions, we will only perform repeats when most reactions submitted with that plate failed to pass our QC. We will then initiate some test runs (straight repeats, repeats with alternative protocol, dilute repeats and/or repeat with higher template volume), to see if we can improve the sequencing results. If results can be improved, we will initiate second runs of all remaining reactions with the best working condition and/or protocol.

For optimum sequencing results and template preparation, please refer to our Sample Submission Guidelines.

For Pre-Defined reactions, please always submit 20 µL of your template in a 1.5 mL flip cap reaction tube or in a well of a semi-skirted 96-well plate. Primer needs to be submitted in a separate 1.5 mL tube or a separate 96-well plate (plate submission only allowed for full-plate service when using a primer mirror plate).

Please note: Our sample processing workflows are fully automated and deviating volume of template or size of sample tubes from our recommended Sample Submission Guidelines, will not be processed.

Please submit 30-100 ng/µL of template when sequencing purified plasmid samples. When submitting low concentration plasmids (EU customers only, Plate or Vario Plate submission required), please submit with a concentration of 10 ng/µL.

For purified and unpurified PCR products, please submit your template in a concentration of 10-50 ng/µL. For short PCR products (<250 bp), we recommend submitting the DNA in a lower concentration (10-25 ng/µL). For longer PCR products, we recommend submitting the samples in a higher concentration (30-50 ng/µL).

Please provide 20 µL of your primer in a separate 1.5 mL flip-cap reaction tube and in a concentration of 10 µM.

For Pre-Mixed reactions, please submit your template and primer mixed in the same 1.5 mL tube or in the same well(s) of a 96-well plate.

Please prepare purified template DNA in a total volume of 10 µL, using 5 µL of template (plasmid concentration range 30-100 ng/µL, purified PCR product concentration range: 10-50 ng/µL) and 5 µL of your primer (stock solution should be at 5 µM).

For Sanger sequencing in tubes and for Vario Sanger sequencing plates, please submit your primer in a 1.5 mL flip-cap reaction tube with a concentration of 10 µM and a volume of 20 µL. This volume is enough for up to 8 reactions, if you would like to have more than 8 reactions processed, please submit a second tube and label it with a second primer barcode. The primer name needs to be modified for the second primer tube and primer barcode (e.g. PrimerX_Aliquot1 for the first tube and PrimerX_Aliquot2 for the second tube). For sequencing in plates and for Vario plates (more than 8 reactions per primer), please submit your primer in a 1.5 mL flip-cap reaction tube with a concentration of 10 µM and a volume of 60 µL. This volume is enough for up to 96 reactions. If you would like to have more than 96 reactions processed, please submit a second tube labelled with a second primer barcode.

Please see the list of our FREE GENEWIZ Universal Primers.

Primer synthesis is available within Sanger sequencing orders by using our “Oligo Retain for Sanger” service. Synthesized primers will be stored at the corresponding sequencing facility for 6 months.

Within these 6 months, your synthesized primer(s) can be used for your Sanger sequencing reactions ordered (Pre-Defined service type) and will be replenished in case the primer stock becomes empty.

Please note: For Express sequence data delivery, please order the corresponding “Oligo Retain for Sanger” by 9am (CET) the same day as sample submission.

To order our “Oligo Retain for Sanger” service, log in to your GENEWIZ account and click Oligo Synthesis > Oligo Retain for Sanger.

Please make dilutions in water or Tris. For best results, do not use Tris-EDTA (TE), as EDTA will inhibit the sequencing reaction. For best sequencing results, please follow our Sample and Submission Guidelines.

Please be sure that your PCR buffer does not contain any EDTA which will interfere with our purification method. For best sequencing results, please follow our Sample and Submission Guidelines.

GENEWIZ provides Sanger sequencing barcode labels to allow handy sample labelling, sample identification, and sample processing within our automated workflows. Our prepaid barcodes can be used as a convenient prepayment option for all our Sanger sequencing services and sample types submitted for sequencing in tubes or plates. 

To order GENEWIZ Sanger sequencing barcodes, please log in to your customer account and navigate to Sanger Sequencing > Order Barcodes and select one of the following:

• Tubes (1.5 mL flip cap reaction tubes)
• Plates (invoiced by full 96-well plate, 1 primer per plate for our Pre-Defined service)
• Flexible Vario plates (reaction-based invoicing, up to 4 primers per well for our Pre-Defined service)

Non-prepaid barcodes are only specified for the corresponding service priority (Express vs Standard) and are not specified for our service types (Pre-Defined vs Pre-Mixed). They can be flexibly used for any of our Sanger sequencing services. As soon as you have some activated non-prepaid barcodes in your CLIMS account and enter your sequencing order, you can determine the service you want to utilize. For sequencing orders using non-prepaid barcodes, invoicing is issued separately for each individual sequencing order after the corresponding sequencing order has been performed in our sequencing lab.

If prepaid barcodes are used, the invoice is issued immediately after the barcodes are activated in your CLIMS account, so that all reactions that are coded with these barcodes are paid at once. To place a prepaid barcode order, you will need to provide us with your payment details directly within the barcode order.

The turnaround time for Sanger sequencing results using our service priority “Standard” is next business day after sample receipt at GENEWIZ. When submitting samples for standard data delivery please use white barcodes.

The turnaround time for Sanger sequencing results using our service priority “Express” depends on whether you submit your samples to GENEWIZ in tubes, plates or Vario plates:

• For samples submitted in tubes or Vario plates, data delivery is next business day by 10am after sample submission. Please note that express turnaround time is only available for purified PCR products and plasmids and with GENEWIZ Logistics available at your location. Please contact us at logistics.europe@genewiz.com for more details.

   

• For samples submitted in plates, data delivery is next business day by 12pm after sample submission. Please note that express turnaround time is only available for purified PCR products and plasmids that are submitted with the Pre-Mixed service type. For the Express service you will furthermore in most cases require DHL Express Return Labels that are provided by GENEWIZ.
  
  

When submitting samples for express data delivery please use blue barcodes. You can either drop off your samples at your GENEWIZ dropbox, at a DHL Express station or hand over samples to a DHL Express driver (individually booked pickup).

 

For UK Customers: Please either use Royal Mail to post your samples to the GENEWIZ lab in Takeley, download a freepost label that can be used to send in the post, or use a courier that collects from your local GENEWIZ dropbox. Standard service is available with a turnaround time of 24 hours from sample receipt at our Takeley facility. London, Cambridge, Oxford and Norwich customers using a GENEWIZ dropbox will receive their Sanger data the next business day from 9am after sample drop-off. Customers requesting PCR purification may take longer, although we will try to process in the same time frame. Please contact us at dnasequk@genewiz.com for more details.

Tube Barcodes per Reaction can be used for Pre-Mixed or Pre-Defined sample types submitted in 1.5 mL tubes. 1 tube barcode corresponds to 1 reaction (1 primer-template combination).

Barcodes for Sequencing in Tubes Pre-Mixed or Pre-Defined Standard:

Your barcode order will be processed promptly in our facility in Leipzig, Germany or in Takeley, UK (for UK customers only).

The required barcodes of your order will be activated in your CLIMS customer account. Together with your ordered barcodes you will be provided with the required shipping supplies for sending your samples to our facility, if needed. After the order has been processed, you will receive an email with the DHL tracking number of your barcode shipment and a description of our GENEWIZ Logistics services.

For UK: Customers that have GENEWIZ barcode stock available at their institution can collect non-prepaid barcodes from the store and activate them onto their account directly. For prepaid barcodes, after you place an order online, collect the batch from the store and send the batch ID and verification code to the UK customer service team (dnasequk@genewiz.com) to get your barcodes activated.

Primer barcodes can be ordered from the Order Barcodes page when purchasing non-prepaid or Pre-Defined barcodes for Tubes, Plates or Vario plates. If you choose not to order primer barcodes, we will send you a defined amount, based on the quantity of non-prepaid or Pre-Defined prepaid barcodes ordered (e.g. with 100 tube reaction barcodes we will ship 20 primer barcodes).

Primer Barcodes:

If you would like to assign barcodes to members of your lab, you can set up a Payment center within your customer account on CLIMS. To get started, log into your customer account and select the Payment center tab at the bottom of the page.

To order sequencing reactions with GENEWIZ, you will first need to order barcodes for one of our sequencing services (please see the “How can I order barcodes?” section). As soon as you have received your barcodes, you can enter an order for one of our three Sanger sequencing service types by clicking on the corresponding icon in your customer account:

Barcodes for Sequencing in Tubes Pre-Mixed or Pre-Defined Standard:

Semi skirted 96-well sequencing plates (Sanger Sequencing Plate service) must be labelled with the correct barcode number that was assigned to the sample plate within the order sheet. Also 1.5 mL primer tubes must be labelled with the barcode number that was assigned to the primer tube within the order sheet.

1.5 mL template tubes (Sanger Sequencing Tubes service) must be labelled with the correct barcode number that was assigned to the corresponding reaction within the order sheet. 1.5 mL primer tubes must be labelled with the barcode number that was assigned to the primer tube within the order sheet.

Semi skirted 96-well sequencing Vario plates (Sanger Sequencing Vario plate service) cannot be labelled with a barcode number (virtual reaction barcodes will be assigned to the different wells). Please label your plate with the order number. For Vario plates, primers should be submitted in 1.5 mL primer tubes and labelled with the barcode number that was assigned to the primer tube within the order sheet.

Barcodes for Sequencing in Plates Pre-Mixed or Pre-Defined:

Within our Pre-Defined Tube service, you can order up to 8 different reactions (with 8 different primers) per template (Volume 20 µL). Please ensure the sample names (DNA template names) are identical for these different reactions. Afterwards, please use the label of the 1^st barcode number assigned to this sample name for the labelling of your template tube. The remaining barcode numbers that were generated in our online portal for this template can be discarded since they are not usable anymore.

Pre-Mixed samples cannot be stored within our automation and are directly discarded after the reactions are completed.

Our facility stores your submitted Pre-Defined templates and primer aliquots for 10 business days after the samples have been received at GENEWIZ. Within these 10 business days, you will be able to submit re-orders with your template and primer.

Please note, that aliquots of 20 µL of primer and 20 µL of template can only be used for a maximum of 8 reactions each. If you already ordered 8 reactions with a primer or template, you would need to submit a new aliquot.

After 10 business days, the quality of primers and templates deteriorates dramatically, which will significantly affect sequencing results. We therefore do not guarantee for high-quality sequencing results after that time period.

Re-orders can be entered for pre-defined samples only. Pre-mixed samples are not stored. For reordering, please select the “Samples at GENEWIZ” check box in your Sanger sequencing order form and provide us with the tracking number of your original corresponding order.

When your barcode order has been processed and the barcodes are ready for courier pickup, you will receive an email that explains your shipping option either via a GENEWIZ dropbox or an individual sample pickup by a DHL Express Courier.

For UK: If your institution has a GENEWIZ dropbox, you may use this for free sample pickup by one of our couriers. Otherwise please ship by Royal Mail in a padded envelop/GENEWIZ shipping case. Find details on our Samples and Logistic Options page available for your location.

Please see the list of our GENEWIZ Universal primers.

When ordering Pre-Defined reactions in tubes, plates, or Vario plates, our system will display a separate column that can be used to select a universal primer for your tube, plate, or well reaction.

Reactions submitted in tubes: “GENEWIZ Primer Selection Tool”

We recommend designing your sequencing primers in a region that is 100 bases upstream of your sequence of interest. If that seems impossible, the closest you want the primer to be to your area of interest is 50-60 bases. Anything closer would risk you are missing a portion of your area of interest. The primers should be about 18-24 bases in length with a Tm of 56-60 degrees Celsius. The GC content should be about 45-55%. For the oligo purity, desalting is all that is needed for sequencing.

After your order has been processed and the corresponding data have been synchronized to your customer account, you can view and download your results from the “My Orders” section of your customer account home page. You can download your data by clicking on “View Results” (green button) for the corresponding order.

The data are provided as .seq and .ab1 files. The .seq file is a text file and can be opened with any program that can view text files (WordPad, Notepad, etc.) If you are having difficulty opening these files, you can change the file extension from .seq to .txt to view it.

The .ab1 file is your chromatogram, and requires chromatogram viewing software to view these files. Please visit Tools for Sequencing Data or several free software programs that are available for viewing trace or chromatogram files.

Please visit Tools for Sequencing Data for several free software programs that are available for viewing trace or chromatogram files.

No priming

No PCR product was generated during the sequencing PCR, which can be due to one or more of the following reasons:

• Too low/high template amount added in the sequencing PCR
• Too low/high primer amount added in the sequencing PCR
• Low quality template (salts, EDTA, phenol contamination) used for the reaction
• Primer does not bind efficiently to template sequence (blocked by e.g. secondary structure near-by the primer binding site)
• Primer not added to sequencing reaction
• Primer has incorrect Tm (we recommend that you use our universal primers or design your own primers that are about 18-24 bases in length with a Tm of 56-60 degrees Celsius)

   

  Poor quality

  Almost no PCR product was generated during the sequencing PCR, which can be due to one or more of the following reasons:

• Too low/high template amount added in the sequencing PCR
• Too low/high primer amount added in the sequencing PCR
• Low quality template (salts, EDTA, phenol contamination) used for the reaction
• Primer does not bind efficiently to template sequence (blocked by e.g. secondary structure near-by the primer binding site)
• Primer has incorrect Tm (we recommend that you use our universal primers or design your own primers that are about 18-24 bases in length with a Tm of 56-60 degrees Celsius)

   

  Non-specific

  More than 1 PCR product (products with different sequences) was generated during the sequencing PCR, which can be due to one or more of the following reasons:

• Primer binds twice / non-specifically to your template sequence
• Primer degradation
• Template added in the sequencing PCR was not a “single-band PCR product”

   

  High background

  Your chromatogram shows a small background (2^nd signal) under your normal peaks. This can be due to one or more of the following reasons:

1. More than 1 PCR product (products with different sequences) was generated during the sequencing PCR, which can have the following reasons:
   • Primer binds twice / non-specifically to your template sequence
   • Template added in the sequencing PCR was not a “single-band PCR product”
   • Primer started to degrade

      

2. Your sequencing reaction was inefficient, leading to the visibility of the background noise.

   (see poor quality)

   Homopolymeric

   After homopolymeric/repetitive regions, the polymerase is often slipping from the template DNA leading to a non-informative / non-specific signal. In this case, we recommend to sequence from the opposite direction.

   Early Termination

   A secondary structure within your template sequence inhibits the polymerase to properly read through your sequence. For pre-defined reactions, we will repeat your sample with our alternative protocol that might help to break the secondary structures.

   Smear

   Sequencing PCR was too efficient. Extremely high PCR product amounts usually lead to an overload of the capillary on the sequencers.

• Very high template amounts added in the sequencing PCR. We will dilute your samples when repeating pre-defined reactions. (For pre-mix reactions, there are no repeats possible. Please resubmit your sample with a lower concentration.)

   

  Spectral pullup

  Sequencing PCR was too efficient. Extremely high PCR product amounts usually lead to an overload of the capillary on the sequencers.

• Very high template amounts added in the sequencing PCR. We will dilute your samples when repeating pre-defined reactions. (For pre-mix reactions, there are no repeats possible. Please resubmit your sample with a lower concentration.)

 

If you do have any further questions regarding your sequencing results, please contact us at Sanger.Europe@genewiz.com and provide us with your project’s GENEWIZ tracking number.

For Pre-Mixed reactions, no repetition can be provided as the end user submits the samples Pre-Mixed (template DNA + primer) and GENEWIZ will only perform the sequencing without storage of the samples. For our Pre-Defined service, if the reaction does not pass our QC in the first sequencing run, our Technical Support team initiates some test runs (straight repeats, repeats with alternative protocol, dilute repeats and/or repeat with higher Template volume) to see if we can improve the sequencing results. If results can be improved, we will initiate second runs of all remaining reactions with the best working condition and/or protocol.

For UK: Pre-Mixed reactions cannot be repeated as the end user submits the samples Pre-Mixed (template DNA + primer) and GENEWIZ will only perform the sequencing without storage of the samples. Repeats of Pre-Defined reactions can be processed upon request. Please contact dnasequk@genewiz.com to speak with a Technical Support representative.

Sanger-EZ is a prepaid, Pre-Mixed service that combines the convenience and sample security of barcoded tubes and the quality of automated Sanger sequencing. With Sanger-EZ, you’ll enjoy the highest level of ease as subsequent Sanger reactions do not require online orders. After you receive your Sanger-EZ tubes from GENEWIZ, simply fill them with your DNA + Primer and send them back to us for sequencing! Our system will autogenerate an order in your account referencing the barcode numbers on the tubes as DNA names.

When our lab receives your Sanger-EZ samples, our system will create a new order (40-xx/50-xx/90-xx) in your GENEWIZ account and will be visible in the “My Orders” section. Results can be viewed here when ready.
You will receive an email with a link to view the results when available.
IMPORTANT: The barcode number of the tube will be used as the DNA/Sample Name when viewing the results. Please make sure to record this number prior to submission.

The Sanger-EZ service is available for purchase within your GENEWIZ customer account. Select the Sanger Sequencing tab on the left and then the bubble labeled “Sanger-EZ”. Sanger-EZ tubes are sold in racks of 96 tubes.

Sanger-EZ utilizes our Pre-Mixed service type. Please submit your DNA + primer in the provided Sanger-EZ tube, according to our Sample Submission Guidelines for our Pre-Mixed service.

1. Prepare your reactions according to our Sample Submission Guidelines for our Pre-Mixed service.
2. Sanger-EZ tubes can be submitted to either a GENEWIZ dropbox or shipped to a GENEWIZ facility. IMPORTANT: if submitting to a “By-Request” GENEWIZ dropbox, a pickup request must be placed to ensure sample pickup by our courier. Pick-up requests are made within your GENEWIZ account under “DROP-OFF AND DELIVERY BOXES”.
3. Ensure to note the barcode number for each reaction. This number will act as the sample name to view results after processing.

Sanger-EZ is offered in both Standard and Express turnaround time.

Unfortunately, due to the automated workflow of Sanger-EZ, repeats are not available. Please contact Technical Support Sanger.Europe@genewiz.com for assistance.

Yes, you can share your Sanger-EZ tubes with colleagues within your institution! This can be done from the “My Sanger-EZ” bubble or tab.

Find the Sanger-EZ batch you would like to share and click “Assign”.

Select 1 or more colleagues and follow instructions to share Sanger-EZ tubes.