The success of automated sequencing critically
depends on having high purity template in
the correct concentration. In case of samples in plates, constant
concentration among wells is also important for the success of sequencings.
i) Plasmid DNA
Preparation
There are many commercial kits
available. Please submit DNA
in deionized water. Do not use
TE to dilute or resuspend the
DNA because EDTA inhibits the
cycle of sequencing reaction.
We recommend using Qiagen miniprep/midiprep
since both methods yield consistent
purity of plasmid DNA for sequencing.
Please provide DNA in the concentration
range of 100ng/µl and in the amount of at least 2 µg.
Extra amount of DNA ensures that
we have enough sample for a re-sequencing
in case the first reaction fails.
If samples' concentrations do
not fall within this range or if you fail to provide us enough
template to do the reaction, the experiment might be delayed.
ii) PCR fragments
Preparation
The DNA is free of contaminants,
unused primers or dNTPs. PCR
templates that do not undergo
any kind of post PCR clean up are not suitable for sequencing and
will yield
unusable sequence data. It is
highly recommended that your
PCR template is first observed on a gel to confirm that there is
a specific product
with the correct size. The Qiagen
Gel extraction kit or PCR cleanup
kit can be used to remove all of the unwanted elements from your
template.
2) Host strains
The host strain can have an impact on the
quality of the template DNA prepared evenusing
the best methods.
DH5-α strains consistently produce
good results. HB101, XL-1 Blue, JM109 and
MV1190
are usually fine but JM101 is often poor.
The growth media you use can also affect
the outcome yields, while LB is usually fine.
3) Quantitation
Sequencers are able to handle a wide range
of DNA concentrations however with very low
amounts of DNA the data quality will be significantly
affected.
Using UV absorbance to quantitate dilute
DNA solutions tends to give widely inaccurate
results.
A good way to quantitate
DNA is to run an aliquot on a minigel and
compare the intensity to the a control of
a known concentration. There
are also concentration ladders that are commercially
available. For each reaction, please provide
10 ng/100 bases, and at least 20 ng/µl
solution in deionized water. Please provide
at least 10 µl for any possible re-sequencing.
Measurement with electrophoresis is recommended rather than spectrophotometer-based measurements.
4) Primers preparation
Primer
Considerations
Primers should be provided in DI water at the required concentration
(see table above).
- High Purity
- Appropriate concentration
- No secondary priming sites
- No mismatches
- A length of 18-25 bases.
- GC% content between 40% and 60%.
- A Tm (melting temperature) between 55°C and 60°C
- No significant hairpins (>3bp)
- Free of salts, EDTA, or other contaminants
Please supply primers at concentration of (10 pmole/µl =60
ng/µl) in deionized water at volume of greater than 20 µl.
Primers supplied by customers should be desalted or purified. Crude
primers generally do not work well for sequencing. We have the following
primers available at no extra charge.
Macrogen Korea #60-24, Gasan-dong
Geumchen-gu
Seoul, Korea
Macrogen Europe Meibergdreef 39
1105 AZ, Amsterdam
the Netherland
Macrogen USA 9700 Great Seneca Hwy
Rockvill, MD, 20850
United States
Macrogen Japan 1-1-1 Sakuragaoka,
Setagaya-ku, Tokyo
156-8502, Japan
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2005-2011 (C) Macrogen Inc. All rights reserved.
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