Bi-directional Sequencing

Bi-directional Sequencing Under this service, we can provide DNA/ plasmid isolation to bi-directional sequencing up to 1500 bases. Customer can also provide us tissue samples, genomic DNA, purified plasmid or PCR products.

Template DNA Information 

• Indicate the size of plasmid DNA on the order form. For large DNA, we use different cycling conditions and longer extension time.
• Indicate the size of the PCR DNA on the order form.  We adjust the sample concentration based on the product size.
• Accurately indicate the DNA concentration.
• If DNA concentration is very low, success of sequencing result will be very less and considered as QC fail.
• Sequencing of any PCR products, need to check for optimal concentration (please go through sample preparation). Need to mention on order sheet, if your DNA sequences contain high GC, repeated sequences, or poly A, so we can modify sequencing reaction conditions. The success of sequencing depends on right information.

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 to avoid confusion. Do not use the same template name more than once.

• Do not make your sample name too long.
• Any combined template and primer names with more than 28 characters will be truncated.
• If you use tubes with screw-on caps, please label both the tube and the cap.

Shipping

• Use proper precaution for shipment to prevent mechanical damage of tubes containing DNA or PCR products. However, DNA may be contaminated and may not generate clean data.

Sample submission

             Sending samples in the right way for our Value Read service. Sending samples according to the requirements below helps us to do our job better and provides you with accurate results.

• Use 1.5 ml safe-lock tubes for your templates and primers or you may send sample in 0.2mL PCR product. Keep 0.2mL PCR tubes in 1.5ml of Eppendorf tube. Wrap entire set-up with par film to prevent any evaporation or leakage
• Do not tape or wrap tubes with Para film. 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:

               Quantify the template concentration via agarose gel or a photometer to ensure accurate results.

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