More primers will be added to this list, please check this website frequently

• Updated on July 1st, 2006

Technical supports for sample preparation and submission

• General Information to submit your sequencing samples

We prefer your samples in 0.2 ml 8-strip PCR tubes, 96-well PCR plates with 8- or 12-strip caps. You also can use common Eppondorf tubes. The template DNA should be dissolved in 10 mM Tris-cl buffer, pH 8.5 (no EDTA!). H2O dissolved DNAs may not be kept longer.
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Generally, The DNA template submitted to us is a doubled amount compared to that needed by one sequencing reaction. For plasmids, 12ul (100-160ng/ul), and for PCR products, 12ul (20-50ng/ul, depending on fragment sizes). See table bellow.

Please label your tube on the SIDE with the corresponding sample ID and fill your Sequencing Sample Order Form(MS Excel format). Cap your tubes tightly with matching caps. Try to provide some cushion around the tubes to avoid any potential damaging during shipping. Send your tubes to us by express mail carriers (Fedex is preferred). Please check Priority Overnight option for timely delivery.

As a reference, the amount of template needed per reaction, please refer to the following table:

For Plasmid DNAs
Because most labs use automated plasmid extration machines to make mini-preparations of plasmids, or use QIAGEN plasmid mini-prep Kits to manually purify plasmids, it has simplified the sequencing sample submission. We make following suggestions for you:

1. Culture a single colony in 3-4ml LB medium with appropriate antibiotics overnight (12 hours, never over 16 hours, The genomic DNA released by dead bacteria may significantly affect the sequencing reaction). Pellet all the bacteria for plasmid extraction;
2. Purify plasmid DNA by QIAGEN Mini-prep Kit, and final elution volume is about 40-50ul;
3. Submit 12ul (about 200ng/ul) of plasmid solution for each sequencing reaction;
4. Submit 5ul of primer x (20ng/ul) each reaction if the primer is not on our Ready4U primer list;
5. Shipping via Fedex overnight delivery

.For DNAs from PCR products

PCR products as templates should be purified using a method that eliminates primers, nucleotides, and extraneous bands. Usually, agarose gel fractionation plus gel extraction kit purification are enough. We suggest you use QIAGEN Quick PCR Preps Kit for primer removal if your product shows only one sharp specific band. After purification, it is important that you verify the presence of only a single PCR product band on a gel.
Accurate quantification of template concentration is especially important for sequencing PCR products. Absorbance on a spectrophotometer at 260nm may not give you an accurate measurement of DNA concentration. We recommend running the sample alongside a DNA Molecular Weight Marker of known concentration on an agarose gel to estimate the sample amount. Lambda DNA MW Marker digested with Eco RI and Hind III is usually used in labs as sample quantification estimation, which has a MW nearly 50kb. If you load 500ng of the MW marker, the amount of 1kb DNA band is around 10ng.
We can process samples submitted in all common size microtubes, although we prefer that samples be submitted in 0.2 ml tubes or 96-well plates designed for PCR with caps. Please don't send samples in regular microtiter plates (i.e.,those designed for immunoassays). Those plates are brittle and sometimes crack during shipment, resulting in sample loss.)

Realgene provides High Quality,Low Cost and Fast Turnaround sequencing service.Our prices for DNA sequencing services:

• Sequencing from cleaned and dried samples: $8.0 /reaction

• Sequencing plus purification: $9.0 /reaction.