Preparing a good template for sanger sequencing

The quality of the DNA template is one of the most critical factors in Sanger sequencing.  Please ensure that the template is at the correct concentration, that it is free of contaminants and dissolved in distilled water (especially no EDTA present), and that the DNA is not degraded.

BRF users are required to follow our guidelines for the amount of sample to submit to the facility.

  • Core prep - please submit a minimum of 15µL

There are several steps for producing good templates

1.  Isolating the DNA

Plasmid Preparation

For plasmid preparation (either large-scale or mini-preps), numerous methods work if you are sufficiently careful to avoid genomic DNA, excess RNA or contaminants.

PCR Products

For PCR products there are two options, depending on the cleanliness of the PCR reaction.  If the PCR product is exceptionally pure, you can use an ultrafiltration device that separates the PCR products from the primers, discarding the latter.  Otherwise, run the PCR products out on a gel and gel-elute the desired band.  Note that the PCR product must be very pure in order to obtain clean sequence.  Extraneous bands may appear low-intensity to you, but could easily ruin the sequence run.  Note also that you must remove all traces of the original PCR primers, as these could produce undesired bands by acting as sequencing primers.

2.  Quantitation of template DNA

If you have enough template DNA, always determine its concentration by UV absorption.  However, most of the time, you cannot reliably quantitate a mini-prep using a spectrophotometer.

Some comments about getting accurate spec readings on DNA

  • Readings below 0.05au require careful blanking in order to ensure accuracy.
  • Many specs cannot be trusted below 0.05au, and most should not be used below about 0.02au.
  • Remember that RNA and free nucleotides also absorb UV, so the sample must be free of these contaminants.
  • Be careful to avoid chromosomal contaminant DNA.  On a restriction digest, it shows up as a smear in the background.  Any visible smear is likely to represent a large percentage of the total DNA present, thus throwing off your measurement.

If you do not have enough to measure spectrophotometrically, then run a small aliquot on a gel and estimate the amount of DNA by comparison with standards of similar size and known concentration.  If you are unable to determine the concentration accurately, you should consider not proceeding until you have enough DNA to measure accurately.

3.  Diluting the template to the desired concentration

After determining the concentration, dilute the template to the correct final concentration using distilled water (please, no TE or other EDTA-containing buffers), and avoid adding any divalent cation (Mg, Ca, Mn).

This information has been drawn from the University of Michigan DNA Sequencing Facility Web Page.