This is a short summary and some highlights of important steps
of dye terminator sequencing of DNA. For more details, see the
manual distributed together with your sequencing kit.
PRE SEQUENCING STEPS
Template DNA
The most crucial aspects of fluorescent DNA sequencing
are the quality and amount of the template DNA in the
sequencing reaction.
PCR fragments
Use as high Ta (annealing temperature) as possible for
the primer pair in the PCR reaction and prolong the
last elongation to 10 min.
The PCR fragments need to be purified in order to remove
excess PCR primers and nucleotides. This is most easily
done with PCR fragment purification kits or enzyme digestion.
If there are two or more bands in the PCR sample, run
it on agarose gel and cut out the band of choice before
purification.
Visually check the amount and quality of the purified
DNA by running a few µl on an agarose gel before
performing the sequencing reaction.
The use of buffers for solving DNA that contain more
than 0.1mM EDTA could significantly reduce the effective
concentration of Mg2+ in the sequencing reaction. The
DNA polymerase can thus be inactivated.
Shorter fragments give more copies per ng DNA. Adjust
the amounts of template DNA in the sequencing reaction
according to the PCR fragments length. Recommended amount
is 3-50ng depending of length (~3ng/100bp).
Please note that we do not recommend sequencing PCR fragments shorter than 150 bases.
Optimal quantity of PCR products (24 cycles reaction):
smaller than 200 bp: 1-3 ng
200-500 bp; 3-10 ng
500-1000 bp; 5-20 ng
1000-2000 bp; 10-40 ng
larger than 2000 bp; 20-50 ng
Plasmid DNA
The best method for preparing templates is the Caesium
Chloride method, but in most cases a miniprep kit gives
excellent results. It is important not to overload the
plasmid columns, especially when performing mini-preparations.
Overloading can result in contaminated DNA. Contaminated
plasmid DNA can be cleaned with phenol/chloroform extraction
followed by ethanol precipitation. Visually check the
amount and quality of the purified DNA by running a
few µl on an agarose gel before performing the
sequencing reaction.
Larger plasmids give fewer copies per ng DNA. Adjust
the amount of template DNA in the sequence reaction
according to plasmid size. Recommended amount is 0.15-0.3µg
for an ordinary vector size (4-8kb). For large plasmids
(over 10kb), use up to 1µg DNA.
Sequencing primer
Poorly designed primers of poor quality will yield little
or no sequence. Therefore you should give careful thought
to primer design and treat your primer solutions with
care.
Try to achieve a melting temperature (Tm) of approximately
55-60°C in 18-24 bases. The primer should preferably
contain around 50% GC. If the GC-content exceeds 55%,
the denaturation of the primer might be incomplete.
Avoid secondary structures and strings of four or more
of one base in the primer sequence.
If the primer solution is exposed to many freeze-thawing
the primers might be degraded. Always make working solutions
from the stock solution.
Use 3.2pmol (3.2µM) primer in the sequence reaction.
If you are sequencing very large clones (e.g. BAC, PAC,
lambda), use 30pmol per reaction.
THE DNA SEQUENCING REACTION
Choose Ta to get high specificity for the
primer. Annealing temperatures ranging between 40-70°C
works fine for the kits. Keep the elongation temperature
at 60°C.
The number of cycles can be increased to yield more
labelled fragments if previous analysis has shown weak
signals but rather good sequence results.
It is possible to dilute the sequencing kits if previous
analysis has shown strong signals and good sequence
results. If you are using version 1.1 or 3.1 BigDyeTM
keep the final volume of 20µl and add appropriated
amount of the dilution buffer that is included in the
kit.
POST-SEQUENCING STEPS
There are different kits to use for removal of unincorporated dye-labelled ddNTPs. These nucleotides will show up as "blobs" if not removed, covering the beginning of the DNA sequence. If you choose to use ethanol precipitation, follow the instructions in the BigDye manual carefully, or use the procedure below. It is important to remove all ethanol with a tip after the centrifugation steps.
Ethanol precipitation (20µl reaction)
Add 2µl 125 mM EDTA, 2 µl 3M NaOAc pH5,3 and 50 µl 100 % EtOH.Incubate on ice 10 min. Centrifuge 10 min. at maximum speed in room temperature.Remove as much of the supernatant as possible. Add 250 µl 70 % EtOH. Vortex briefly.Centrifuge 5 min.Remove as much of the supernatant as possible.Dry the pellet (protect from light).
Updated
by Annika Eriksson
May 28, 2008.
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