Download Automated DNA Sequencing

Host Strain The host strain used for template preparation can impact template quality. One host
Variability strain may produce better sequencing results for a specific template than another.
If you plan to use a commercial template preparation kit, contact the vendor for
information about host strains that work well with that kit.
A good source of information relating to host strain effects can be found in the
QIAGEN Guide to Template Preparation and DNA Sequencing (2nd edition). Contact
your local QIAGEN office (http://www.qiagen.com/qiagenww.html) to obtain a copy of
this guide.
Determining DNA The following methods can be used to examine DNA quality:
Quality ♦ Agarose gel electrophoresis
Purified DNA should run as a single band on an agarose gel.
Note
♦
Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear.
Spectrophotometry
The A260/A280 ratio should be 1.7–1.9. Smaller ratios usually indicate
contamination by protein or organic chemicals.
Agarose gels reveal the presence of contaminating DNAs and RNAs, but not proteins.
Spectrophotometry can indicate the presence of protein contamination, but not DNA
and RNA contamination. These methods should be used together to get the most
information about your DNA template before sequencing.
Note
RNA contamination up to 1 µg can be tolerated, but it will affect DNA quantitation
greatly.
Neither of these methods shows the presence of contaminating salts that can cause
noisy data. If you suspect that your DNA is contaminated with salt, remove the salt
before sequencing. The most efficient method for salt removal is ultrafiltration with a
Centricon-100 column (see page 3-12). Spin columns and ethanol precipitation can
also be used (see page 3-33).
Cleaning Up Dirty A “dirty” template preparation sometimes can be cleaned up with one of the following
Templates methods:
♦
Purify the DNA by ultrafiltration. Use Centricon-100 Micro-Concentrator columns
(see “Preparing PCR Products for Sequencing” on page 3-12).
♦
Purify by extraction:
Step
Action
1
Extract the DNA twice with 1 volume of chloroform or chloroform:isoamyl alcohol
(24:1 v/v).
2
Add 0.16 volumes of 5M NaCl and 1 total volume of 13% PEG.
3
Incubate on ice for 20 minutes, then centrifuge at maximum speed in a
microcentrifuge at 2–6 °C for 20 minutes.
4
Rinse the pellet twice with 70% ethanol.
5
Dry the pellet in a vacuum centrifuge for 3–5 minutes or to dryness.
3-16 Performing DNA Sequencing Reactions