Preparation of Template for Automated DNA Sequencing

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

There are several steps in producing good template:

1. Isolating the DNA

   • For plasmid preparations (either large-scale or mini-preps), numerous methods work IF you are sufficiently careful to avoid genomic DNA, excess RNA or contaminants. A recent poll of our users showed that a large fraction of them use Qiagen products with good success, but other manufacturer's products may work as well (Note: if you use Promega 'Wizard' kits, make sure you use the Wizard SV; do not use earlier versions). You can use Cesium Chloride preps, but make very sure there is no Cs or EDTA present in the final sample. You may need to do an extra ethanol precipitation for this reason.

   • 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 from either Amicon or Millipore 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 sequencing 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!. See Why can't I use a spectrophotometer to check my miniprep?.

   Some comments about getting accurate spec readings on DNA:

   • Readings below 0.05 AU require careful blanking in order to ensure accuracy.
   • Many specs cannot be trusted below 0.05 AU, and most should not be used below about 0.02 AU.
   • 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 desired concentration

   After determining the concentration, dilute the template to the correct final concentration (see table) using *distilled water* (please no TE or other EDTA-containing buffer), and avoid adding any divalent cations (i.e. Mg, Ca, Mn).

If you are looking for information on how to sequence BAC DNA directly, you should consult the following page: Sequencing from Large-Insert Clones