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Sample Preparation Guidelines

The preparation of various samples for ordering a sequencing service depends on the type of sample being submitted, the type of sequencing chemistry being ordered and the additional services being requested. Prepare your samples based on the guidelines below.

Concentration of Stock Solutions

Prepare stock solutions of purified/prepared templates and primers in ddH2O based on the size of the template being sequenced. Use these stock solutions to  assemble samples to be submitted in tubes or plates. These guidelines do not apply to samples for Ready2Load Sequencing service.

Table 1: Concentrations of the stock solutions of primers and prepared templates

Template Type Size (kilo basepairs) [Template] (ng/μL) [Primer] (pmol/μL)
PCR Products, Purified 0.100–0.300 10–20 2–10
PCR Products, Purified 0.301–1.000 20–40 2–10
PCR Products, Purified >1.000 40–60 2–10
Plasmid, Prepared < 1 .000 30 - 60 2–10
Plasmid, Prepared 1.0 << 6 .0 60-150 2–10
Plasmid, Prepared 6.0 << 20.0 150-250 2–10
Large Contructs (BAC), Prepared >20 250–500 10–15

Note that the concentration of the Template is in ng/μL while the concentration of Primers is in pmol/μL.


Volumes of Stock Solutions for Assembling Samples

The volumes of the Sample stock solution(s) and the Primer solution(s) depend on the type of the sequencing services required. Ensure that the stock solutions are prepared according to the guidelines in table 1 before proceeding to section below.

'Premixed' Samples:

When you submit your samples as 'premixed', ensure that the primer is mixed with your purified/prepared template. After assembling the Templates and Primers as described in the table 1 and prepare your sample by mixing the following volumes of the two stock solutions.

Table 2: Assembling Premixed samples
Component Volume (μL)
Template DNA 5
Primer 5
Total (Sample) 10

Note that only the premixed samples must be submitted for ShortSeq plate and the SimpleSeq Premixed sequencing services. Ensure that the samples for 'ShortSeq plates' or 'SimpleSeq Kits - Premixed' are prepared based on the guidelines set up here for 'Premixed' samples.

'Templates Only' Samples

When you do not have the standard or custom primers handy or ready for use, you can submit 'Templates Only' Samples. For the primer required in this sequencing reaction, you can

  • Use one of our >75 universal primers to be added to your sample before sequencing (free), or
  • Order the synthesis of a custom primer that will then be used for sequencing your samples and subsequently stored in our facility for your future sequencing orders

Table 3: Assembling 'Templates Only' Samples.

Sequencing chemistry Template Stock solution — Volume (μL)
First Reaction Each additional reaction
Standard Read 10 5
Power Read 10 10

 

'Templates and Primers' Samples:

When you submit 'Templates and Primers' Samples, our sequencing technicians will mix the primer(s) with your sample(s) prior to sequencing. The ability to send primers and templates separately allows you to submit samples for multiple sequencing reactions or order additional services such as PCR purification/DNA Preparation for your template sample. The following table provides guidelines for the submission of prepared templates and primers. Use the concentration of templates and primers in table 1 to prepare the stock solutions. When you submit 'Templates and Primers' samples:  

  • You must submit your primers and templates in separate containers,
  • Concentrations of template DNA and Primers must adhere to the recommended values (see table 1).
  • Provide sufficient quantities of samples and primers (see table below). You can order multiple reactions for a template submitted in 1 tube or a single well of the 96-well plate. Please note that the volume requirements vary for standard and power read sequencing services.
  • Use barcode labels (optional) on the primer and sample containers,
  • Indicated on the tube sequencing (check box) or plate sequencing (mention in notes) order editor if you want the sent primer to be stored at our facility for your future orders. On request, these primers will be stored in our facility for 1 month or till they are exhausted, which ever is earlier.

Table 4: Assembling 'Templates and Primers' samples

Sequencing chemistry Template Stock solution — Volume (μL) Primer Stock Solution — Volume (μL)
First Reaction Each additional reaction First Reaction Each additional reaction
Standard Read 10 5 10 5
Power Read 12 12 12 6

Samples Requiring 'Additional Services':

When your sample requires further preparation or purification prior to sequencing, the following conditions should be met:

  • The template(s)/Sample(s) and primers(when enclosed) should be submitted in separate containers
  • The volume of the sample based on its type should meet the following requirements (see table).

Table 5: Assembling samples which require additional services

Sample Type Type of service Sample Solution — Volume (μL) [Primer] (pmol/μL) Primer(s) stock solution — Volume (μL)
Crude PCR Products (>100 bp) PCR Purification 15–30 (Entire Reaction Volume) 2–10 10 (5 for each addition rxn)
Bacterial Cultures RCA Plasmid preparation 10 (Glycerol stock recommended) 2–10 10 (5 for each addition rxn)

Preparation of 'Ready2Load' Samples

Ready2Load sequencing services is provided for samples that have a single requirement: to be run on the sequencing gel. Samples being submitted as Ready2Load samples must meet the following requirements:

  • The BigDye v3.1 reaction must be completed.
  • The purification steps that remove unincorporated dye molecules must have been completed.
  • It is recommended that these samples are sent as dry pellets.

Note that the Ready2Load plates are processed using an abbreviated protocol which typically have a read length of 500–700 bp.

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Types of Services and accepted Samples

We have tailored our DNA sequencing services using the Sanger method considering the diverse natures of the projects and requirements of the researchers. Researchers can send different types of samples in different formats with or with out including the primers or requiring additional services on the samples.

Sample/Services Feature Description
 Services Provided The following services are provided:
  • DNA Sequencing using the Sanger Method#
    • for Research samples
    • for Clinical Samples
  • PCR Product purification, and
  • DNA Preparation
  • Fragment Analysis - Ready2Load (click here for sample submission guidelines for Fragment Analysis)
Accepted Sample  types The following types of templates are accepted:
  • Plasmids
    • as Clones
    • Purified or crude
  • PCR Products
    • Crude
    • Purified
  • BACs/Fosmids/Cosmids
  • Post BigDye samples (Read2Load samples)
  • Clinical Samples (See CLIA/CAP page)
Sequencing Chemistries Standard Read - Suitable for most standard templates requiring read lengths of upto 700 bps
Power Read - Suitable for templates with hard to read segments in the sequence. These can include:
  • GC rich segments
  • Sequences with nucleotide repeats
  • shRNA contructs
  • Sequences with secondary structures
Accepted Sample containers These include:
  • Custom Tubes (with or without* free barcodes from Eurofins Genomics)
    • 1.5 mL Centrifuge tubes
    • 2.0 mL centrifuge tubes
  • Barcoded SimpleSeq Tubes
  • Custom 96-well plates
  • Barcoded 96-well plates from Prepaid Plate kits

 

Sequencing Services provided for various Sample Types based on the inclusion of Sequencing primers

Each of the sequencing reaction requires a Sequencing primer that is specific to the template being read. We provide the most widely used sequencing primers for no additional cost. However, if you do need custom primers, our DNA synthesis lab will synthesize and deliver the same to the sequencing lab with remarkable turnaround times.

Sample Types Description
Premixed Submit the template of interest premixed with sequencing primer to receive the results from a standard read in the shortest time.
Templates Only Submit only your template. You can choose from a wide range of services for samples submitted as templates only. These include:
  • Use of over 75 free standard sequencing primers
  • Synthesis of custom primers specifically for your use
  • Use of stored primers (Custom primers are stored for 6 months after their synthesis at our facility)
  • Upgrade to power read for difficult to read template segments.*
Templates and Primers When you send your sample templates and primers in separate containers, we can provide the following additional services:
  • Mix the primer supplied by you and then sequence the template
  • Store the sent/enclosed primer for upto a month (you need to tick the box during the tube sequencing order or incate in notes in the plate sequencing order)
  • Upgrade to power read.*
  • Provide additional services such as DNA preparation (for Plasmids) or PCR Purifications (for PCR Products).*
Custom

Research Services: Whether you need the sequence of a large construct such as BACs or Fosmids, crude PCR product, or you just need the sequencing gel run after having performed the BigDye® reaction, we have a service tailored for your need. If you do not find something for your specific need, please contact your sales representative or write to us at genomicsUS@eurofins.com.

Clinical Research Services: Our CLIA and CAP accredited sequencing lab provides services suited to your specific needs. Contact our CLIA specialist for more information.

#For more details on Next Generation Sequencing Services, click here.
*Additional charges apply.

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Tube Format

<48 samples 

Submit Tubes

All tube types are submitted on the same webpage except SimpleSeq Premixed which does not require online submission.

Barcoded Tubes

  • 1.5 – 2.0 mL Centrifuge tubes, with or without barcodes.
  • Eurofins Genomics provides free barcode labels for use with templates and primers that are submitted for DNA sequencing services.
  • Submitting online: Enter the barcode on the tube submission page.
 

Non-Barcoded Tubes

  • 1.5 – 2.0 mL Centrifuge tubes, with or without barcodes.
  • Submitting online: leave the barcode field empty and proceed to checkout.

8 Strip PCR Tubes

  • Variety of sizes.
  • If submitting less than 8, please cut the strip and save the remaining tubes
  • Submitting online: Write barcode on the side of your tubes with a sharpie marker. 8-strip barcodes are automatically generated and displayed on the online order page using your initials and a number.
   

SimpleSeq Tubes

  • Pre-labeled
  • Tube size is 0.75 mL
  • All tubes provided with kit
  • Fastest turnaround time, along with normal barcoded tubes.
  • Submitting online: Type in the barcode found on side of tube on the order page.

 


 

Plate Format

>48 samples

Submit Standard and Prepaid Plates

Order Prepaid Plates

Submit ShortSeq

Submit Ready2Load

 

Sequencing Standard 96-well Plate

  • Pre-labeled
  • Tube size is 0.75 mL
  • All tubes provided with kit
  • Fastest turnaround time, along with normal barcoded tubes.
  • Submitting online: Type in the barcode found on side of tube on the order page.

Prepaid Plate Kit

  • Pre-labeled
  • Tube size is 0.75 mL
  • All tubes provided with kit
  • Fastest turnaround time, along with normal barcoded tubes.
  • Submitting online: Type in the barcode found on side of tube on the order page.

ShortSeq Plate

  • Pre-labeled
  • Tube size is 0.75 mL
  • All tubes provided with kit
  • Fastest turnaround time, along with normal barcoded tubes.
  • Submitting online: Type in the barcode found on side of tube on the order page.

Ready2Load Plate

  • Pre-labeled
  • Tube size is 0.75 mL
  • All tubes provided with kit
  • Fastest turnaround time, along with normal barcoded tubes.
  • Submitting online: Type in the barcode found on side of tube on the order page.
     

 

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All information on shipping and/or physically submitting samples can be found on the shipping samples page.

Download results

Go to your Order History. Next, click the order to expand and reveal the download link. Click the link to download a zip file of the results. To visit the Order History page, click one of the many links located throughout the website, including in the header beside your account name.

When looking at the order history page, each type of order is color coded by its product line, so it is easy to find the order you want. For example, sequencing orders are marked by a small blue icon, oligo orders are marked by an orange icon, and so forth. Please note that sequencing results are only saved for a limited amount of time. If you are trying to download very old results, it may not be available online but may be archived and available by manual retrieval. Contact us for more information.

Guidelines for Overnight Sequencing Service

Overnight Sequencing service from Eurofins Genomics allows you to advance your research project in an uninterrupted manner within a limited budget. To avail the Overnight Sequencing service from Eurofins Genomics, your order/samples must meet the following requirements:

  • The samples are shipped using 'Overnight Shipping' service
  • The order must require less than 192 sequencing reactions
  • This services applies only to samples requiring 'Standard Read' sequencing chemistry (this does not include other services such as power reads, BAC, PCR purification or RCA)
  • Samples must be supplied in one of the following containers
    • 1.5-mL or 2-mL centrifuge tubes with barcode labels* pasted on them
    • 96-well plates with a free barcode label pasted at the bottom of the plate
    • Use barcode-labeled tubes or semi-skirt Plates supplied by Eurofins Genomics, e.g., as tubes from SimpleSeq Kits or 96-well plates from the PrePaid plate Kits
    • Use of Screw Caps or Sealing using parafilms prevents cross contamination, but can extend the turnaround time.

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