Sample quality
DNA concentration and quality is vital to ensure successful sequencing. We strongly recommend that all customers check the concentration of their samples prior to dispatch.To self- check please measure the OD (Optical Density) 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. This may 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. A high value at OD 320 indicates a contaminant. These should be ideally 0.0. The OD 230/260 ratio should be above 0.6.

We also recommend ensuring the quality of your DNA by running your sample on an agarose gel or taking advantage of our DNA quality checking service

Sample volume
For sample numbers less than 46 submit 10 μl [minimum] per sample at the following concentrations:

Samples MUST be cleaned-up prior to submission and should be sealed & clearly labeled prior to dispatch (either with Parafilm^® (tubes) or seals (plates)). It is preferable if samples submitted in tubes are sent to the laboratory in 1.5 ml tubes.

For half or full plates use semi-skirted 96-well plate. For half plates, pipette samples into odd numbered columns (i.e. 1,3,5,7,9 and 11) ENSURE WELL A1 REMAINS EMPTY. In each well, send 2 μl of template at the recommended concentration (see table above), plus 1 μl of primer at 3.2 pMol/μl. If you seek to take advantage of our offer to resequence your samples without delay in the event of an unforeseen problem (see Terms and Conditions) then you will need to send duplicate plates to the laboratory for analysis as an insurance measure.

A sequencing standard will be run with every group of samples analysed, and well A1 should be left empty. Customers should clean-up their samples prior to submission to the laboratory and supply OD 260/280 ratios along with sample submission information

Results will be e-mailed to customers once analyses are completed.

Can template DNA be sent for sequencing in EDTA containing buffer (TE,TBE)?
Please do not send DNA in EDTA containing buffer. EDTA binds bivalent cations such as Mg2+ that are essential for the function of Taq polymerase. Your DNA is stable in ultrapure water or Tris-HCl at room temperature for several days. This can be increased to several weeks if dried down via ethanol precipitation. If you want to add Tris-EDTA to your DNA, please take an aliquot from the sample prep and send it to us.

Primers
We will sequence your DNA using the primer(s) of your choice, input the requisite information on the online sample submission form. Select from our universal primers or forward a custom primer of your design [5 μl per reaction should be supplied at a concentration of 3.2 pmol/μl]. Tubes MUST be sealed (wrapped with Parafilm or equivalent). For more than one sequencing reaction send the appropriate volume of primer in a single tube. Primers should be freshly prepared and, for the avoidance of doubt, should not have been subjected to multiple freeze-thaw cycles.

Primer information should be included with the samples transferred to the laboratory along with submission summary information.

Template clean-up
For clean-up we recommend the following procedures:

PCR Template DNA
• Qiagen QIAquick^® PCR purification kit
• Qiagen QIAquick^® gel extraction kit
• Promega Wizard^® PCR clean-up kit

Plasmid template DNA

• Qiagen QIAprep^®spin plasmid kit
• Promega Wizard^® Miniprep kit

A final ethanol precipitation step is recommended after use of the Promega Wizard^® kit.

Host Strains for plasmid preparations:

DNA template quality may be compromised dependent on the bacterial strain from which the DNA is isolated. ABI have evaluated a number of host strains and make the following recommendations:

Additional information sources:

Applied Biosystems Guide to Automated DNA sequencing chemistry
http://www.genome.ou.edu/big_dyes_plasmid.html