Template DNA Requirements

The quality of the template is the main factor influencing read length:

• Template DNA must be free of EtOH, EDTA, RNA, salts, genomic DNA and proteins
• Please use distilled water for elution
• Plasmid DNA should mainly be present in covalently closed circular form (Recommendation: silica membrane-based spin column kits)
• PCR products need to appear as a single band in an agarose gel and have to be purified from reaction buffer, primers and nucleotides

Sample preparation

The required amount of DNA template for one sequencing / primer walking step is listed below. Please provide enough DNA to cover the whole sequence. Make sure to add some extra DNA to cover re-analysis if necessary.

 

Template DNA	Volume/~650 bp	Concentration
Primer Walking projects
Plasmids*
<10kb	10 µl	100 ng/µl
PCR products
500 - 1,000 bp	10 µl	20 ng/µl
1,000 - 2,000 bp	10 µl	40 ng/µl
Sequencing primer	10 µl	5 pmol/µl

Primer design and synthesis are included - see separate list of universal primers, already synthesised

 

HiQSeq projects
Plasmids*
<10kb	20 µl	100 ng/µl
PCR products
500 - 1,000 bp	20 µl	20 ng/µl
1,000 - 2,000 bp	20 µl	40 ng/µl
Sequencing primer	10 µl	5 pmol/µl

Primer design and synthesis are included - - see separate list of universal primers, already synthesised

 

*) For sequencing of plasmids larger than 10 kb, high CG content DNA or DNA with difficult DNA motifs (such as secondary structure) please contact CyberGene

 

Customer Primer Requirements

If you include your own primers please consider the following in order to make the sequencing project run as fast as possible:

• Length of 18 - 24 bases
• The annealing temperature should be at least 52°C
• GC-content of at least 40 - 50%
• 3'-end should be G or C
• No self hybridisation (primer dimer, loops) or binding to several sites on the template
• One HPLC product (i.e. not containing truncated, shorter oligo fragments)
• Deprotected (standard part of the oligo synthesis process)
• Without modifications (fluorophore or others)
• Free of salts and other contaminants

CyberGene Quality Document Q07-114-e-01