Single Pass Sequencing

Single pass sequencing means one directional sequencing either of any of 5 prime or 3 prime site of either forward or reverse primer of gene of interest or plasmid. The partial short read sequencing only tell us the accuracy of priming while for complete gene information always advisable to complete gene sequencing by bi directional approach of sequencing.

Under this service, we can provide all biological samples (DNA/ plasmid, direct PCR products etc) isolation to single pass sequence   up to 800 bases. You can also provide us tissue samples, crude DNA, plasmid or PCR products.

Template DNA Information 

• Indicate 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 time.
• Indicate the size of the PCR amplicon on the order form.  We adjust the sample concentration based on the product size.
• Accurately indicate the DNA concentration.
• If your DNA/PCR amplicon concentration or plasmid look like degraded, it will appear to be QC fail to us and we will communicate with you.
• For sequencing of PCR product, DNA concentration needs to be check after purification as one of the crucial step for good quality of sequencing.
• Indicate 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 on 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 more than once.
• 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 instruction to avoid any error.

• Use 5 ml safe-lock tubesfor your templates and primers
• Do not tape or wraptubes with parafilm. Safe-lock tubes offer perfect sealing and evaporation protection
• Use a water resistant marker for any additional labeling of template and primer tubes

Sample preparation

Use the following concentration and volume for your samples:

Note: Quantify the template concentration via agarose gel or a photometer to ensure accurate results before send sample to Eurofins.

Premixed samples (a mixture of template and primer):

• Templates should consist of 15 µl purified DNA with either of the concentrations given in the table above.
• Add 2 µl of primer with a concentration of 10 pmol/µl (10 µM).
• Ensure that the total volume of your premixed sample is 17 µl

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/µlprimer 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
• Concentration of primers with wobble bases must be calculated according to the following formula: n^X xConc_Primer
  n = number of bases within a wobble according to IUPC code, X = number of wobbles within the primer sequence. [e.g. 1 V (AGC) = 3¹ x 10 pmol/µl; 2 V (AGC) (AGC) = 3² x 10 pmol/µl]