The right answers to frequently asked questions
Find the answers to all our products and services by clicking the links below.
Products & Services
How long are prepaid products valid?
All type of prepaid barcode labels, kits and coupon codes are valid for 5 years. The validitiy of unused barcodes are expiring after 5 years.
You can ask for an email notification before your barcodes expire in your personal preferences.
What are Tube Labels - when / why do I need it?
Tube Labels are free barcode labels used for error free labelling and identification of your samples in tubes for all kind of Sequencing services. They allow fast processing your sequencing order. You can track the usage status, the usage date as well as the user of the lables under "May Sanger Kits & Labels".
Tube Labels can be ordered online and are free of charge.
When should I use primer barcodes?
If you send your own primers for sequencing, use the yellow primer barcodes to identify them.
An overview of which labels were used when and by whom can be found under "My Sanger Kits & Labels".
Primer barcodes can be ordered online for free.
How long will my sequencing data be stored?
We will keep your data 6 months after finishing your reads in your personal account. During this time, you can download and save your data.
How long do you store samples and primers?
For all services, DNA samples are stored for 4 weeks.
Synthesised primers are stored for 4 weeks free of charge.
Storage of synthesised primers for one year is available at additional cost.
Enclosed primers are stored for 10 days free of charge.
Storage of enclosed primers for 6 months is available at additional cost.
During this time it is possible to use the DNA and primer for further reactions.
Premixed samples (DNA mixed with the primer) and pre-cycled samples for the Ready2Load service will be discarded after the order is finished and the data submitted.
Can I send shRNA constructs or other DNA templates with probable secondary structures?
Yes, we accept templates with more difficult sequence structures.
Can you sequence phage or genomic DNA?
Please contact our Customer Support to get the best recommendation for your request.
When do I get my sequencing results?
Overview of delivery times for the majority of our Sanger services:
||Data delivery next
business day until
|Data delivery next
business day until
||12 pm CET
||6 pm CET
||1 pm CET
||6 pm CET
|PlateSeq Mix NXP
||1 pm CET
||6 pm CET
||6 pm CET
||12 pm CET
||6 pm CET
||6 pm CET
||6 pm CET
Specified delivery times are mainly depending on sample arrival time in the lab.
For many European cities we are providing coordinated pick-up and transport services using Eurofins DropBoxes ensuring on time delivery in the lab.
However, these logistics services may also be subject to force majeure or unpredictable changes caused by e.g. strikes, accidents, storms, floods. In such events, Eurofins Genomics cannot guarantee the stated data delivery time and is not responsible for delays caused by logistics services.
Additionally, our general terms and conditions (GTCs) apply.
How to order
How can I order your DNA sequencing service?
Please use our online shop for every Sanger Sequencing product and service. In the order menu you find all links to the respective sequencing order pages.
You will find all necessary information regarding sample preparation for each sequencing service under in the sample submission tab of the respective service.
Where should I send my sequencing samples?
Eurofins Genomics has already installed hundreds of DropBoxes in Europe for free sample pick-up. Your nearest DropBox location with pick-up time is displayed in your account when you are logged in.
In case there is no DropBox near you, you can of course send your samples by post or arrange for them to be delivered by another delivery company. Please send Sanger sequencing samples to the addresss which is shown to you during checkout.
Can I order more than one read per sample?
Yes, indepenent on the sample type, more reactions per sample can be specified. Just follow the instruction on the order page.
How many plates per order can I send?
Up to 15 plates can be uploaded / specified at once and saved in you cart. There is no limitation on the number of plates per order you can checkout.
Can I re-order sequencing runs?
You can order new sequencing reactions from stored samples within 4 weeks no matter if you have sent the samples in tubes or plates.
If you have sent the samples in tube format, just use the same order page and type in the initials of the respective sample. The system prepopulates all stored samples with these initials.
If you want to re-order reactions from a few samples originally sent in plates, you find the option "Re-Order from stored plates". Select the stored plate and specify the new reactions for the respective samples.
Under "My samples & primers" you can check the expiration date of your samples and primers.
Can I order additional services when using prepaid barcodes?
With prepaid barcodes, you can order additional services such as primer synthesis DNA mini preparations. You will be billed separately for this.
How should I send my sequencing samples?
You will find all information about sample format, template and primer concentration as well as shipping information for each of our sequencing services in the respective "Sample Submission" tab.
How should I determine the concentration of my samples?
Please measure the OD with a spectrophotometer at 260 nm and 280 nm. The OD 260 values should be in the range of 0.05 to 0.8 to give reproducible and reliable results. The OD 260/280 ratio should be about 1.8. Values lower than 1.6 and higher than 2.0 indicate contaminants in the sample that interfere with the determination of the concentration and might inhibit the sequencing reaction.
If possible, we recommend measuring the ODs at 230 nm and 320 nm, too. A high value at OD 320 indicates a contaminant. This should be ideally 0.0. The OD 230/260 ratio should be lower than 0.6.
We also recommend ensuring the quality of your DNA by running your sample on an agarose gel.
Which purification method should I use?
For plasmids, we recommend the use of commercially available
plasmid mini scale preparation kits employing spin columns. From
our experience, repeating the washing step will improve the quality
of the DNA and therefore improve the sequencing results. Please do
not send DNA prepared with alkaline lysis methods or with the
boiling method (Holmes and Quigley, 1981) without column
purification. For PCR products please use commercially available
PCR purification spin column kits.
Can I send my DNA samples in Tris-EDTA (TE)?
No, please don't.
EDTA binds bivalent cations such as Mg2+ that are essential for the Taq polymerase. Your DNA is stable in double distilled water or Tris-HCl at room temperature for several days or even longer if it is dried down.
Should I submit my DNA samples cooled?
This is not necessary; DNA is stable in water at room
temperature for several days. Just send your samples either in
Tris-HCl, distilled water or air dried. Please, do not use EDTA as
this will inhibit the Taq polymerase.
Can I send unpurified PCR products or clones?
Yes, for all sequencing services we offer template preparation and PCR product purification as an additional service.
Which E.coli strain should I use?
Successful isolation of plasmid DNA is possible from most E.coli
strains, though the strain which is used can have a significant
influence on the quality of the purified DNA. Host strains such as
DH10b, DH5 alpha, XL10 Gold and TOP10 normally result in high
quality DNA in combination with many commercially available plasmid
DNA isolation kits and are therefore ideal for the propagation of
plasmids to be sequenced. Lower quality DNA is derived from strains
producing large amounts of carbohydrates, which are released during
lysis and inhibit enzyme activity. Examples are HB101 and its
derivatives such as TG1 and the JM100 series. Also strains with
medium or high levels of endonuclease activity like HB101, JM101 or
BL21 generate DNA of lower quality, hence a proteinase K treatment
should be considered.
Do I need to clean up my PCR product if there is only one band visible?
Yes, primers and dNTPs must be removed from the product. The primers would produce a mixed sequence (5% contamination with a primer results in unreadable mixed sequences!) and the dNTPs would interfere with the dNTP/ddNTP concentration in the sequencing reaction. We recommend purification by using commercially available PCR spin column kits. Alternatively we can perform the PCR product purification for you as additional service.
I see unspecific bands in my PCR reaction. Do I need to gel
purify my product?
If you want to employ one of the PCR primers in the sequencing
reaction you must elute the product of interest from an agarose
gel. Otherwise, a mixed sequence will be produced as the primer is
most likely to bind both the specific and unspecific products. If
you employ a specific nested primer in the sequencing reaction, it
might work. However, producing only one product in the PCR reaction
is the best guarantee for a good sequence. In almost all cases,
improving the PCR conditions (specific primer design, higher
annealing temperature and/or lower primer concentration) helps to
avoid unspecific products.
Primers & More
Which standard primers do you offer?
80 different standard primers are available for your DNA sequencing order. for free Under "My samples & primers" you see the list of standard primers.
Can I send my own primers for sequencing ?
Yes of course. Just specify your enclosed primers in your sequencing order with the primer name. The primers should be sent in a solution and concentration specified in the respective "Sample Submission" pages. Please use a primer barcode to identify your primer.
Can you synthesise a primer for me? How do I order the synthesis?
Yes, we can synthesise your sequencing primer in our oligo production. Just specify your primer for synthesis in your sequencing order with the respective name and sequence.
How should I send my primers and how much primer do you need?
Please find information about primer format, primer concentration and primer shipment information for each of our sequencing services in the respective "Sample Submission" tabs.
Can I pay for primer synthesis using barcodes?
The primer synthesis along with Eurofins sequencing services can be payed with prepaid barcodes.
What are IUPAC bases? Which results will I receive?
DNA base call identification is based on the nomenclature system
issued by the International Union of Pure and Applied Chemistry
(IUPAC). At positions of ambiguity, the following IUPAC codes are
|G/T = K
||C/T = Y
||A/C/G = V
|A/G = R
||A/C = M
||C/T/G/ = B
|G/C = S
||A/G/T = D
||A/C/G/T = N
|A/T = W
||A/C/T = H
What are Q20 (Q30 / Q40) bases?
Eurofins Genomics is using a quality base calling software which examines the peaks around each base call to assign a quality score (Q) to each base call. Quality scores (Q) range from 4 to about 60, with higher values corresponding to higher quality. The quality scores are logarithmically linked to error probabilities, as shown in the following table:
|Quality Score||Probability of a wrong base call||Accuracy of a base call|
||1 in 10
||1 in 100
||1 in 1.000
||1 in 10.000
||1 in 100.000
Which sequencing method do you use?
For all our Sanger Sequencing Services we are using the cycle sequencing technology (dideoxy chain termination / cycle sequencing) on ABI 3730XL sequencing machines.
Could you explain the cycle sequencing technique?
Cycle sequencing is a modification of the traditional Sanger sequencing method. The components are DNA, primer, heat resistant DNA polymerase, 4 dNTPs, 4 ddNTPs (dideoxy terminator nucleotides) fluorescently labelled with four different dyes and buffer containing Mg++ and K+. The single primer binds to the complementary DNA strand and is extended in a linear mode.
This extension continues until by chance and depending on the complementary base a particular ddNTP is incorporated. Because of the latter's dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. Thus at the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are generated.
Stoichiometric manipulation of the reaction components ensure that the fragments of every possible length starting from n+1 say 2000 bases are generated with n being the number of bases in the primer. The key difference between the traditional Sanger method and cycle sequencing is the employment of a thermo stable DNA polymerase. The advantage of using such a polymerase, is that the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions.
After a post sequencing reaction cleanup, the samples are electro kinetically injected into the array of 96-capillary sequencers. The negatively charged fragments migrate toward the anode by size, the smallest ones move fastest. Their tagged ddNTP terminators can be read as the fragment's base sequence. A laser beam excites these dye molecules as the fragments reach a detection window, producing fluorescent signals that are collected from all 96-capillaries at once, spectrally separated and focused onto a CCD (charge coupled device) camera. Sophisticated optical and electronic devices produce a colour readout that is translated with the help of sequence analysis software into a sequence.
What is the quality report?
The quality report is the trace file in *.pdf format in which the quality value of each single base is shown in colour code below each single peak. Different colours represent the four specified quality ranges.
Are there special features regarding the standard DIN EN ISO 17025?
Under the conditions listed below, only simplified test reports may be provided.
Subject: Simplified test reports according to DIN EN ISO 170252018, (Section 7.8)
Testing Laboratory (Accreditation): Eurofins Genomics Europe Applied Genomics GmbH (D-PL-13372, ) Eurofins Genomics Europe Sequencing GmbH (D-PL-17038)
For accredited examinations at these two companies, test results may only be reported in simplified form, i. Requirements of the standard DIN EN ISO 17025: 2018 (section 7.8) are not fully applicable to this type of transfer of results.
These results can therefore also be reported as short test reports, electronically generated result reports, Excel spreadsheets or as result files.
If you have any questions, please contact the Customer Care Team.
How can I view the .ab1 trace files
There are many softwares on the market which you can use for testing or buying.
For your convenience you can download and install our GATCViewer software for free.
What does "DONE_QV_NOT_MET" mean?
In order to ensure that the sequencing quality is not affected by a technical failure, an internal quality control is performed with each sequencing run. In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths.
If you see "DONE_QV_NOT_MET" as a result of your sequencing reaction, it means that the internal quality control has passed, but some sample quality values have not been met. (e.g. sample or primer concentration or volume, sequence design or structure etc.).
Whole Plasmid Sequencing
How does whole plasmid sequencing compare to Sanger sequencing?
There are pros and cons of every sequencing method. Sanger sequencing and Oxford Nanopore sequencing (ONT) are both methods for determining the sequence of nucleotides in a piece of DNA. Typically Sanger is consider the most accurate method for short-read sequencing and NGS is better for long-read sequencing.
Overall, the choice between Sanger sequencing and ONT will depend on the specific needs of the application. Both methods have their strengths and limitations, and the appropriate method will depend on factors such as the length and quality of the DNA sample, the desired level of accuracy, and the cost and availability of the necessary equipment.
What is the coverage?
It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length plasmids and how many, if any, degraded.
A coverage exceeding approximately 20x indicates a highly accurate consensus sequence.
Is there a minimum number of samples I must submit?
Any number of samples is welcome. There are no minimums or limitations.
Can you sequence my mixture of plasmids?
You can send it and we can sequence it, but we cannot predict or promise the analysis outcome. The customer would take on the risk of these orders.
Do you offer a prepaid solution for whole plasmid sequencing?
Great news! We are actively working on bringing you a prepaid solution, which will be available very soon!
Can I also sent amplikons and large insert DNA for the Whole Plasmid Sequencing service?
Unfortunately not. Currently, we can only accept plasmids. However, we do offer a special service for amplicons and large DNA inserts, which is available for your use. Learn more >>
Can I send my samples without the free barcode?
Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!
Can I use my local dropbox to ship samples for Whole Plasmid Sequencing?
Yes of course.
If you don't know if there is a dropbox close by, use our dropbox location page to find out. Please click here >>
Will you also deliver the FASTQ data?
Yes, FASTQ files are delivered for every project.
Troubleshooting guide for failed samples
There are several reasons why your sample might have failed:
- The starting material did not consist of circular DNA.
In our library preparation protocol, circular DNA is required as the starting material. If linear DNA is provided, it will result in sample fragmentation and subsequently lead to poor or no results.
- Insufficient DNA concentration in prepared samples.
The primary reason for this issue is the use of a Nanodrop device to measure DNA concentration. We strongly recommend employing a Qubit or a similar method instead.
- Presence of mixed plasmid species and/or fragmented genomic DNA or fragmented plasmids within the samples.
You may detect signs of this failure mode through a wide range of read lengths reported in the raw read length histogram.
To interpret your results you can also have a look at our troubleshooting guide. Click here >>
Which buffer should I use to send my samples?
Please send your samples in nuclease-free water or elution buffer.
Please do not use a buffer containing EDTA.
Will you repeat sequencing of my sample if the sequencing fails?
For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.
What is the turnaround time for whole plasmid sequencing?
We will sequence your plasmids typically within 1 business day.
For plasmids > 25 kb please except a turnaround time of up to 5 business days.