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Template Submission Requirements
 for DNA Sequencing Orders


Plasmid DNA Preparation

Required Palsmid concentration: Plasmid 0.2ug/ul,
Concentration at 5 ul/reaction; thus about 1ug of plasmid per reaction

The quality of template DNA is extremely important to obtain good sequence data. The following methodologies are recommended for preparing plasmid DNA for automated sequencing: Cessium chloride (CsCl) banding, Qiagen plasmid kit, Wizard plus plasmid kit, and other commercial kits available. Magnesium ions are essential for DNA polymerase activity. Template DNA and primer should be resuspended in water or a buffer containing no more than 0.1 mM EDTA. Introduction of large amounts of EDTA in template DNA or primer will result in weak signals or short reads. The optimal concentration of the template should be at 0.2 ug/ul.

PCR Product Preparation

The purity of the PCR product is very crucial for obtaining good sequence data. Any PCR primers and/or dNTPs carryover in the PCR product will adversely affect the quality of the sequence data. If the PCR product has a unique band, it can be purified by using size exclusion method, like PCR purification kit from Qiagen and Pharmacia. If the PCR product has more than one bands, the PCR product should be run on an agarose gel and the band that to be sequenced isolated from the gel. The band can be purified by using the kit from Centricon or Qiagen. The purified PCR products can be quantified either on the gel or spectrophotometer.

Recommended DNA Template and Primer Quantities

  PCR Product   Amount   Concentration
  10-500 bp   150 ng   20 ng/ul
  500-1000 bp   200 ng   20 ng/ul
  1000-2000 bp   300 ng   20 ng/ul
  Single Stranded   600 ng   100 ng/ul
  Double Stranded   1.5 ug   200 ng/ul
  Cosmid, BAC, P1   4 ug   200 ng/ul
  Custom Primer   100 picomoles   10 picomoles/ul


For primer extension with results of 1100 bp sequence data per reaction; we need 5-10ul of primers with the concentration of 10 picomol/1ul of primers.

Custom Primers
If you want Bionexus to synthesize the primers for your sequencing:

 (1) Please DO NOT mix primer with your DNA samples.
Please keep them in separate tubes for Q.C. purposes.

(2) Recommended Scale for Custom primers: For most PCR and sequencing needs, only a minute amount of oligo is needed. For example, the majority of sequencing protocols call for <10 picomoles of primer. Most PCR reactions are done using 0.1-0.5 uM primer. For a 20 uL PCR reaction, 0.5 uM primer is equal to 10 picomoles. For an average 25-mer oligonucleotide, 1 OD260 unit is equal to about 4 nanomoles, or enough primer to do estimated 400 PCR or sequencing reactions.



CONTACT US to order your DNA Sequencing Services today...

Select from one of the following links:
DNA Sequencing Order Form
List of Free Universal Primer Sequences

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