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

• Use either our PlateSeq Kit DNA for purified DNA or our PlateSeq Kit Mix for premixed samples
• Alternatively you can use the 96well FrameStar® PCR plate* from our plate accessories
• Plates with purified DNA may contain plasmids and PCR products
• 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 use fully skirted PCR plates and label the plates with our PlateSeq Labels
• Ship samples at ambient temperature to our sequencing lab

Quantify the template concentration via agarose gel or a photometer to ensure accurate results. Plates with dried DNA samples are also accepted: Ensure that samples are dried in a PCR cycler or incubator to avoid over-drying .

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 either our PlateSeq Kit for unpurified PCR products or the 96well FrameStar® PCR plate* of our plate accessories
• Concentration must be normalised across 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

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

• Use either our PlateSeq 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
• Cover the plate with a lid and loosely wrap with cellophane
• Incubate plate at 37°C for 8-12 hours (overnight)
• 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
• Cover the plate loosely with a lid and incubate at 37°C for 8-12 hours (over night)
• 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 plate with your order number and plate name on the plate frame
• Freeze the plate at - 80°C
• Ship your glycerol cultures on sufficient dry ice to prevent sample decay

*FrameStar® is covered by one or more of the following US patents or their foreign counterparts, owned by Eppendorf AG: US Patent Nos. 7,347, 977 and 6,340,589. FrameStar® is a registered trademark owned by 4titude® Ltd.

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 and can be re-used during this time
• Enclose your own sequencing primers
  You can send us your sequencing primers in 1.5ml tubes along with your samples by using our Primer Barcodes*
• 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); 5 µl of primer volume is required for every additional sequencing reaction
• 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 V (AGC) (AGC) = 3² x 10 pmol/µl]

 *: Purchased Tube Labels can still be used to identify enclosed primers

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

Data storage:

• Secure online archive of all selected sequence data for 6 months
  (For orders that are processed in Ebersberg, the data is stored for 3 months)

When to choose the Power Read Upgrade

In case your samples are >30kbp and/or your samples contain difficult-to-read stretches, select the Power Read Upgrade.

We will perform special cycling conditions or chemistry to achieve best sequencing results. A free repetition is included. This service requires a longer processing time.

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 is run with each plate (for that purpose well H12 should be kept empty, otherwise a quality control is not possible). In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths. If these parameters fall below a certain percentage across the plate an additional manual check is performed. If a technical failure is confirmed by this manual check, the plate is getting re-sequenced.