I. DNA template submission 

A) Samples in tubes:   Please clearly label each tube, preferably on top. If one shipment contains samples for multiple work orders (requests), please ensure that tubes are labeled uniquely, so we can correctly assign and process them. You can also use different tube colors for different requests and/or place them in separate plastic bags/containers.

B) Samples in 8-strip PCR tubes:   This submission format is preferable if the number of samples in a request exceeds 20, but is under 48 to facilitate sample processing. Label your tubes on the sides with the sample number (labeling of every other tube or even first and last in a strip is sufficient). If multiple requests are included in a single shipment, please make sure that the labeling is unique.

C) Samples in 96-well plates (plates with round or conical well shapes are preferable): You can submit any number of samples in this format, especially when the number of sequencing reactions for each sample is limited to one or two. The sample arrangement must be in column/vertical format (A01, B01...H01) to comply with our downstream processing.

See section III below for the recommendations on the amount of DNA templates needed for the optimal sequencing results.

II. Primer/s submission 

A) Primers already pre-mixed with DNA templates:   You can submit your DNA templates with primers already pre-mixed. We recommend that you add 1 µl of 5 µM ( = 5 pmol/µl) primer per well or equivalent. Using lesser amounts of primer is not recommended and using more is not harmful, but unnecessary.

B) Primers submitted separately: We recommend that you dilute your sequencing primer to 5 µM (5 pmol/µl) in 10 mM Tris, pH 7-8. Under these storage conditions primers are very stable and can be re-used multiple times. The shelf-life of primers stored in water (especially at room temperature) is much shorter and they will need to be re-synthesized frequently.

C) Common sequencing primers: Wyzer Biosciences provides many common sequencing primers free of charge. A current list of common primers is located under Common Sequencing Primers link. Please review this list with the recommendations and caution therein. We will expand this list as needed and requested.

D) Primers to be designed and synthesized by Wyzer Biosciences: If requested, we can design and synthesize primers to meet your sequencing needs. These primers will be stored, unless otherwise advised, and can be re-used in your future requests, saving time and money. We use in-house developed and/or commercial software to design primers to assure that they comply with accepted specifications for sequencing primer design criteria.

III. Amount of DNA template 

The total amount of DNA template needed for sequencing depends on the type of DNA (PCR, plasmid, cosmid or BAC) and the size of fragment to be sequenced. If you send pre-mixed DNAs with primers, we recommend that you use at least 50 ng, but no more than 1000 ng for plasmids in the size range 3-30 kbp per tube or well. The optimal amount is 100-250 ng/reaction. For PCR fragments, regardless of the size, use 5 to 500 ng per tube/well. The optimum amount is 25 ng/reaction. Using less or significantly more of the template will result in sub-optimal or even no data.

For cosmids (typically ~ 40 kbp) or BACs (typically 100-150 kbp), use minimally 1000 ng of DNA per tube/well. Adjust the amount accordingly, if multiple reactions are required.

The table below provides the approximate number of reactions and amount of DNA needed to fully complete sequencing, with double-stranded coverage, for different length inserts. For plasmids, we assume that the total size is in the range 3-20 kbp. We also assume that each sequencing reaction read yields 600-800 bases of usable sequence, and the template is of good quality. We use 250 ng for plasmids and 25 ng for PCR for the calculations.

Insert Size (Kbp)	# of Seq Reactions (minimum)	ng of Plasmid DNA (minimum/optimal)	ng of PCR Fragment (minimum/optimal)
0.2 - 0.8	2	100/500	10/50
0.8 - 1.6	4	200/1000	20/100
1.6 - 2.4	6	300/1500	30/150
2.4 - 3.2	8	400/2000	40/200
3.2 - 4.0	10	500/2500	50/250
4.0 - 4.8	12	600/3000	60/300
4.8 - 5.6	14	700/3500	70/350
5.6 - 6.4	16	800/4000	80/400
6.4 - 7.2	18	900/4500	90/450

We strongly recommend that your templates be re-suspended in 10 mM Tris, pH 7-8. Please remember that EDTA (as for example in TE buffer = 10 mM Tris, 1 mM EDTA, pH 8) interferes with sequencing, although a low amount, 0.1 mM or less, is tolerated. These conditions assure that your templates are stable for a long time under most storage conditions. Templates stored in water deteriorate within a few weeks, which may pose problems, if additional sequencing on the same sample is required in the future. We appreciate that you advise us on the type of storage solution you use, particularly if your samples are in water or if the buffer contains EDTA.

Occasionally, reaction failures do occur (e.g. non-optimal primer; ABI machine malfunction; difficult template...). Therefore, it is advisable to submit 20-25% more template DNA than the minimum amount recommended above.

IV. Quality and Quantitation of DNA 

The quality of your DNA prep is the single most important factor in getting good DNA sequencing results. Please be advised that many salts typically used in the laboratory, at concentrations < 25 mM interfere very little with the DNA sequencing process. However, at concentrations > 50 mM they may eliminate the sequencing signal entirely. The presence of other contaminants may also interfere with the sequencing process, so please make sure your DNA samples are pure or contain minimal amounts of interfering agents. Please refer to publication (1) for more details.

If you are confident that your template preparation does not contain RNA, other DNA or compounds absorbing in 230-300 nm range, then using a spectrophotometer to estimate DNA concentration is a very quick and reliable method. Just read the absorption of your sample at 260 nm (in a 1-cm or equivalent cuvette) and apply the following factors, if you diluted your sample, please remember to include it in your calculations:
      50 x OD₂₆₀ of the Sample x Dilution Factor = Concentration of dsDNA (mg/ml)
For ssDNA, this equation is:
      33 x OD₂₆₀ of the Sample x Dilution Factor = Concentration of ssDNA (mg/ml)
For RNA, you should use:
      40 x OD₂₆₀ of the Sample x Dilution Factor = Concentration of RNA (mg/ml)
However, if your DNA samples contain RNA, genomic DNA or other compounds absorbing in 230-300 nm range, we recommend you use gel electrophoresis (e.g. E-gels or Flash gels) or other suitable methods for more accurate determination of your sample concentrations.

(1) Jan Kieleczawa: Effects of Various Reagents on DNA Sequencing Process. in J.Kieleczawa (ed). DNA Sequencing: Optimizing the Process and Analysis. 2005, 11-26, Jones & Bartlett.