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:
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. Elute in distilled water or in
1 mM Tris, if desired. Do not elute in TE, as the EDTA can interfere
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.
It is not necessary to linearize your plasmid before sequencing.
- 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.
See more about how to sequence PCR products on
Quantitation of template DNA
If you have enough template DNA, always determine its concentration by UV absorption.
However, very often, 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.
- Be suspicious! If your yield of DNA seems unusually good, it is probably
too good to be true.
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. Actually, it is a good idea to do this
even if you have performed a spec measurement, in order to cross-check yourself.
If you are unable to determine the concentration accurately, you should consider
not proceeding until you have enough DNA to measure accurately.
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