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  Sample Preparation

General Information about Templates and Primers

All templates should to be checked for quantity and quality before being submitted to SeqXcel for analysis. Poor quality DNA results in:

• A weak signal with greatly reduced read length
• High background peaks or “noise”
• Unreadable sequence data

The most common contaminants are salts, bacterial proteins, cell wall carbohydrates, and organic solvents (phenol, ethanol, propanol, etc.).

Purified DNA templates must be submitted in sterile, deionized H2O or 10 mM Tris (pH 8.0, no EDTA).

DNA Template Purification

We recommend Qiagen DNA purification kits for both plasmids (Qiagen cat.# 27104) and PCR products (Qiagen cat.# 28104). If you are using other commercially-available kits, please consult the manufacturers to ensure they yield DNA acceptable for DNA sequencing.

For larger DNA templates, such as bacterial artificial chromosomes (BACs), cesium chloride (CsCl2) bands or DNA purified by “alkaline lysis” method followed by an extra phenol extraction is recommended. If CsCl2 bands are submitted, make sure there is no CsCl2 or EDTA remaining in the sample.

Kits that will provide DNA of acceptable purity are available for BACs and bacterial genomic DNA (Qiagen cat.#10243).

Checking concentration and purity of DNA template

Using a spectrophotometer, measure your templates absorbance at 260 and 280 nm. The A260/A280 ratio for DNA should be greater than 1.8.

Calculate the concentration of your double-stranded DNA sample from A260 as follows:

dsDNA concentration (ng/µ
L) = A260 X Dilution Factor X 50 ng/µ

Calculate the concentration of your single-stranded DNA sample from A260 as follows:

ssDNA concentration (ng/µ
L) = A260 X Dilution Factor X 30 ng/µL

PCR Products

For PCR products, make sure all traces of the original PCR primers are removed! Do not give us samples straight out of the thermal cycler. Excess primers, salts, or Taq polymerase, interfere with the sequencing reaction.

If the PCR product is a single band, a PCR purification kit, will suffice in separating the PCR products from the primers.

If you have more than one PCR product in your reaction, you will need to separate the products on an agarose gel and excise the band you wish to sequence. Several gel extraction kits for PCR products are commercially available.

Gel purification of PCR products generally produces the best sequence data.

PCR Products and ExoSAP-IT® treatment

PCR products that have been treated with ExoSAP-IT® (USB Corp.) can be submitted for DNA sequencing. Only PCR reactions with a single product yield good sequencing data. Low intensity extraneous bands could easily cause the sequence run to fail.

The ExoSAP-IT® treatment must be mentioned on the order form and be accompanied by a photo of the gel or an estimation of the amount of DNA in the sample.

Recommendations for Primers

Primers should be desalted, but do not require HPLC-purification.

Primers should be resuspended in sterile, deionized H2O or 10 mM Tris (pH 8.0, no EDTA) and have the following properties:

• 18-25 base pairs long
• Tm of 55 to 60oC.
• GC content no greater than 55%
• Does not form primer dimers
• Binds only to one site on the template.

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