Single Pass Sequencing

Single-pass sequencing involves sequencing in one direction, either the 5' or 3' end, using the forward or reverse primer of the gene of interest (GOI) or plasmid.

This method provides partial short-read sequencing to check priming accuracy. For complete gene information, bi-directional sequencing is recommended.

Eurofins Genomics With single-pass sequencing, we can sequence up to 800 bases of plasmids or shorter PCR amplicons.

Check out the DNA Template and sample preparation guidelines added below for best results.

Template DNA Information 

• Mention the size of plasmid DNA on the order form. For complex (GC-rich, Secondary structure) target gene sequencing, we use different cycling conditions and longer extension times.
• Mention the size of the PCR amplicon on the order form.  We adjust the sample concentration based on the product size.
• Right DNA concentration is crucial for the sequencing run (please check our order form).
• If your DNA/PCR amplicon concentration or plasmid looks like degraded or smeared, it will appear to be QC fail to us and we will communicate it to you.
• For sequencing of PCR product, DNA concentration needs to be checked after purification as one of the crucial steps for good quality sequencing.
• Mention if your DNA sequences contain high GC, repeated sequences, or poly A, so we can modify sequencing reaction conditions. 

Sample Name Clarification 

• The name of the sample on the tube and the order form should be identical.
• The name of the template DNA should be different in every order. Do not use the same template name.
• Do not make your sample name too long.
• The name should not be more than 18 characters.
• If you use tubes with screw caps, please label both on tube and cap.

Shipping

• Use proper protection for your shipment to prevent sample tubes from breaking. We will try our best to recover samples from cracked tubes. However, DNA may be contaminated and may not generate clean data.
• When shipping DNA samples for overnight delivery, dry ice is not necessary unless your shipping policy requires it. Room temperature shipping also reduces your shipping cost.

Sample Submission

Please follow the instructions to avoid any errors.

• Use 1.5 ml safe-lock tubes for your templates and primers.
• Use parafilm to wrap the tube to prevent leakage or contamination. However, Safe-lock tubes offer perfect sealing and evaporation protection.
• Use a water-resistant marker labeling of the template and primer tubes.

Sample preparation Guidelines

Use the following concentration and volume for your samples:

Note: Quantity and quality should be checked appropriately before shipping to Eurofins. (concentration mentioned in our sample preparation guidelines)

Sequencing primers

Optimum primer conditions

• Primers must not contain phosphorylation or fluorescent dyes
• The optimum primer length is between 16-25 bases
• The primer melting temperature (Tm) should be 50 - 62°C
• The GC content of the primer should be 35-60%
• Ideally one G or C should be located at the 3' primer end.
• The number of 3' Gs or Cs should not exceed 2 Gs or Cs
• If possible, avoid >3 identical bases in a row in the sequence.

Primer concentration and volume

• Exactly 10 pmol/µl primer concentration is required per sequencing reaction.
• Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl); 5 µl of    primer volume is required for every additional sequencing reaction