DNA Sequencing Guidelines and Tips

If you are submitting your first sample, please contact the lab for assistance.

We request all of our customers to submit sequencing samples containing:

  • the template, primer, and water
  • already combined into a .2 ml tube (available at the Core Facility for no charge)
  • in 6 ul total volume. (Total reaction volume must be 6 UL)
  • a DNA sequencing form (Word) (PDF)

The two most important factors to receiving good sequencing results are:

  • TEMPLATE CONCENTRATIOn
  • TEMPLATE PURITY

TEMPLATE CONCENTRATION:

Providing the correct DNA template concentration depends on careful quantitation by agarose gel electrophoresis in parallel with appropriate standards.

Template quantities per reaction:

  • PCR products 30-70 ng
  • ssDNA ~50 ng
  • BAC DNA 1600 ng
  • dsDNA (for plasmids < 10 kb) 200-300 ng

For dsDNA we require 200-300 ng (~.1 pmole) of template per reaction for templates no longer than 10kb. Plasmid of larger sizes may require more than 300ng. Contact the DNA Lab for information for on large plasmids.

For PCR products we require 30-50 ng. For PCR products that are ~1-2 kb or larger, you may need to use 50-70 ng. Contact the DNA Lab for help determining the amount.

For BAC DNA use approximately 1600 ng (size dependent). Please contact the DNA Lab for help before submitting your samples.

TEMPLATE PURITY:

In order for the DNA Sequencing reaction to function optimally, the template must be very clean. This includes removal of any PCR primers that may be present as well as any cellular debris.

There are several kits on the market which yield good quality DNA.

For plasmid DNA, Qiagen and Wizard minipreps give good results. Other methods give less reproducible results. Centricon and Qiagen work well for removal of PCR primers.

In addition, plasmids purified by "midiprep" and maxiprep" methods work better for sequencing than do "minipreps". The DNA produced is cleaner and, therefore, gives better sequencing data.

Template can be in water, TE, or TE/10 [10 mM TrisHC1, 0.1 mm NaEDTA].

HOST STRAIN:

The bacterial host strain used for template preparation can effect the quality of the sequencing. There is variability of sequence quality between different templates grown in different cell lines.

PRIMERS:

Use 1.6 pmoles of primer per reaction. (BAC DNA: 50 pmoles primer)

Please make sure that there is a primer binding site on your template.

You can go to IDT's website to use their OligoAnalyzer to check for primer dimers, hairpins, and Tms.

Check out our Primer design tips.