Ultimate Sanger Sequencing Service

The Comfort Upgrade includes:

• Quality control of the incoming DNA
• Concentration adjustment of all purified DNA
• Individual reaction conditions according to performed quality control and sample specifications
• Special chemistry if applicable
• Manual analysis of the sequencing result
• Repetition of failed sequencing runs with adjusted sample volume, reaction conditions and chemistry as applicable

Sample submission and preparation for Plasmid DNA, purified PCR products and premixed samples (mixture of DNA and primers)

• Use our PlateSeq Ultimate Kit for purified DNA and premixed samples
• Alternatively you can use the 96well PCR plate from our accessories or your own 96well plates
• Plates with purified DNA may contain plasmids and PCR products
• Template size should not vary by more than a factor of 3
• Template concentration must be normalised across the plate*
• One plate position should be kept free for internal quality control
• DNA samples should be sent liquid in a total volume of 15 µl
• Seal your plates using 8-cap strips to prevent material loss
• If you are using your own plates, please label the plates with our PlateSeq Labels
• Ship samples at ambient temperature to our sequencing lab in Ebersberg

Quantify the template concentration via agarose gel or a photometer to ensure accurate results. 

* As an alternative to the information in the table, the optional concentration adjustment allows the DNA concentration to be varied and adjusted across the plate. Nevertheless, the DNA concentration should not deviate by more than a factor of 3. The concentration is determined randomly to provide consistent sequencing conditions for the plate.

Premixed samples (mixture of DNA and primer):

• Templates should consist of 15 µl purified DNA with either of the concentrations given in above table
• Add 2 µl of primer with a concentration of 10 pmol/µl
• Ensure that the total volume of your premixed sample is 17 µl

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Sample submission and preparation for unpurified PCR products

• Use  our PlateSeq Ultimate Kit for unpurified PCR products
• Alternatively you can use the 96well PCR plate from our accessories or your own 96well plates
• The DNA concentration should not vary by more than a factor of 3; The values ​​in the following table can serve as a guide. The concentration is then randomly measured after cleaning to determine consistent sequencing conditions for the plate.
• PCR product size should not vary by more than a factor of 3
• One plate position should be kept free for internal quality control
• PCR products should be sent liquid in a total volume of 15 µl
• Seal your plates using 8-cap strips to prevent material loss
• If you are using your own plates, please label the plates with our PlateSeq Labels
• Ship samples at ambient temperature to our sequencing lab in Ebersberg

Quantify the template concentration via agarose gel or a photometer to ensure accurate results.

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Sample submission and preparation for plasmid clones as stab culture in soft agar

• Use either our PlateSeq Ultimate Kit for plasmid clones or our agar plates with appropriate antibiotic
• Use sterile toothpicks to pick single colonies from your petri dish and inoculate a single well with one colony
• Seal the plate air permeable and incubate for 8–12 hours (over might) at 37 °C
• If you are using your own plates, please label the plates with our PlateSeq Labels
• Seal the plate with an adhesive plastic foil and ship your stab cultures at ambient temperature to us

Sample submission and preparation for plasmid clones as freeze glycerol cultures

• Only use transparent 96well microtiter plates with a total volume of 350 µl/well
• Fill each well with 200 µl of liquid medium (e.g. LB-medium) including the appropriate antibiotic and add 40 µl glycerol (final glycerol concentration: 10-20%)
• Ensure that liquid cultures are sent only in glycerol!
• Use sterile toothpicks to pick single colonies from your petri dish and inoculate a single well with one colony; or transfer already arrayed clones from a storage glycerol plate to a freshly prepared 96well plate using a multi-channel pipette
• Seal the plate air permeable and incubate for 8–12 hours (over might) at 37 °C
• Verify that the plate surface is dry before you manually seal the plate tightly with an adhesive plastic foil to prevent material loss
• Label the plates with our PlateSeq Labels
• Freeze the plate at - 80 °C
• Ship your glycerol cultures on sufficient dry ice to prevent sample decay

Use the following concentration and volume for your samples:

Quantify the template concentration via agarose gel or a photometer to ensure accurate results.

Premixed samples (a mixture of template and primer):

• Templates should consist of 15 µl purified DNA with either of the concentrations given in the table above
• Add 2 µl of primer with a concentration of 10 pmol/µl (10 µM)
• Ensure that the total volume of your premixed sample is 17 µl

 
Please send your sample tubes according to the requirements below:

• Use 1.5 ml safe-lock tubes for your templates and primers
• Do not tape or wrap tubes with parafilm. Safe-lock tubes offer perfect sealing and evaporation protection
• Label your sample tube with one of your barcode labels. Affix the label horizontally

Benefit from the greatest flexibility to handle your sequencing primers.

Thanks to our DNA synthesis lab, bioinformatic know how and internal IT solutions you can choose how to provide us with your sequencing primers:

• Select from standard primers on stock
  Select and manage your favourite standard primers from more than 80 different validated standard primers
• Order specific sequencing primers
  Specific primers can be ordered directly with your sequencing order. These primers are stored for 4 weeks (storage for one year on request) and can be re-used during this time
• Enclose your own sequencing primers
  You can send us your sequencing primers in separate 1.5ml tubes along with your samples. Use our free Primer Barcodes for this. Enclosed primers are strored for 10 days.
• Premix primer with DNA template
  Sequencing primers can be added directly to your DNA template and sent to us as premixed sample.

Please consider optimal primer conditions, concentration and volume as described below.

Optimum primer conditions:

• Primers must not contain phosphorylation or fluorescent dyes
• The optimum primer length is between 16-25 bases
• The primer melting temperature (Tm) should be 50 - 62°C
• The GC content of the primer should be 35-60%
• Ideally one G or C should be located at the 3' primer end
• The number of 3' Gs or Cs should not exceed 2 Gs or Cs
• If possible, avoid >3 identical bases in a row in the sequence

Primer concentration and volume:

• Exactly 10 pmol/µl primer concentration is required per sequencing reaction
• Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl)*
• Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer

n = number of bases within a wobble according to IUPC code, X = number of wobbles within the primer sequence. [e.g. 1 V (AGC) = 3¹ x 10 pmol/µl; 2 Y (CT) (CT) = 2² x 10 pmol/µl; 1 N (AGCT) = 4¹ x 10 pmol/µl ]

*Sufficient for up to 200 reactions. 30 μl are needed for up to 350 reactions. For more reactions, the primer must be entered several times and with a new name (e.g. indexed)

Please send your samples to:

Eurofins Genomics
Sequencing Lab
Anzingerstraße 7a
DE - 85560 Ebersberg

Use one of our shipping options to send your samples via mail or courier. Make sure to pack your samples safely.

DNA and primer storage:

• Storage of template DNA for 4 weeks
• Storage of synthesised primers for 4 weeks free of charge
• Storage of synthesised primers for one year at additional cost
• Storage of enclosed primers for 10 days

Data storage:

• Secure online archive of all selected sequence data for 6 months
  

Definition of technical failure

A technical failure is defined as a chemical or technical (IT, machines) defect in our lab. 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.

For that purpose, well H12 should be kept empty for samples in plates, otherwise a quality control is not possible.

If above parameters fall below a certain percentage, an additional manual check is performed. If a technical failure is confirmed by this manual check, the sequencing run is getting repeated.