PlateSeq Service - The Swiss Army Knife

Great flexibility based on your requirements

Anything you want to sequence in 96well plates can be done with our PlateSeq Service.

• Solutions for different sample types
  You can provide us with purified DNA, plasmid clones, crude PCR products or premixed samples
• Flexible payment options
  Prepay with our PlateSeq Kits or pay once you have the results
• Flexible sample amounts and number of reads
  Define up to 4 sequencing reactions per sample for a minimum of 48 samples per plate
• Solutions for plate labelling and sealing
  Select from a variety of plate accessories such as PCR plates, agar plates or plate seals, for shipping your samples

The benefits of our PlateSeq Service at a glance: 

• Define up to 4 reactions per sample, per plate
• High quality read lengths of up to 1,100 bases
• Free Q20, Q30 or Q40 monitored single strand data available
• DNA preparation and PCR purification as additional option
• Remaining DNA can be sent back upon request
• Results are available from next day after samples received
• Overnight sequencing available with our PlateSeq Kit Mix ON
• Different sequencing result files can be selected per order
• Secure online archive of sequence data for 100 days
• Option for Power Read Upgrade* for complex sequences
• Free one time repetition in case of technical failure**
• Plates may contain different types of purified DNA samples
• Storage of template DNA and ordered primers for 4 weeks
• Primer storage for one year, on request
• Easy re-order option from complete plates or single samples

*In case the sequences of your samples contain difficult-to-read stretches, select the Power Read Upgrade. We will perform special cycling conditions or chemistry to achive best sequencing results. A free repetition is included.

** 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.

Sending samples the right way for our PlateSeq Service

Sending samples according to the requirements below helps us to do our job better and provides you with accurate results

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

• Use either our PlateSeq Kit for purified DNA or premixed samples or 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
• Label the plate with your order number and plate name on the plate frame if you are using your own plates
• 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
• Label the plate with your order number and plate name on the plate frame if you are using your own plates
• 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)
• Label the plate with your order number and plate name on the plate frame if you are using your own plates
• 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

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Sequencing primers

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: n^X x Conc_Primer
  
  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]

*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.