Protocols and Applications Guide (US Letter Size) - Promega

||||||||| 9DNA Purification
These authors amplified and characterized a putative
epoxide hydrolase gene from the jewel wasp Nasonia
vitripennis. PCR fragments were amplified from genomic
DNA, purified from gels using the Wizard® SV Gel and
PCR Clean-Up System and then subcloned into the
pGEM®-T Easy Vector. The plasmid DNA was purified
using the PureYield Midiprep System. Linearized
plasmids were used for in vitro transcription of RNA for
use in RNA interference experiments.
PubMed Number: 18579785
C. PureYield Plasmid Maxiprep System
As with the PureYield Plasmid Midiprep System, the
PureYield Plasmid Maxiprep System (Cat.# A2392,
A2393) is designed to isolate high-quality plasmid DNA
for use in eukaryotic transfection and cell-free expression
experiments. The system provides a rapid method for
purification using a silica membrane column. Plasmid DNA
can be purified in approximately 60 minutes, greatly
reducing the time spent on purification compared to silica
resin or other membrane column methods.
Like the other PureYield Plasmid Systems, the maxiprep
system also incorporates a unique Endotoxin Removal
Wash, designed to remove substantial amounts of protein,
RNA and endotoxin contaminants from purified plasmid
DNA, and improve the robustness of sensitive applications
such as eukaryotic transfection, in vitro transcription and
cell-free expression. Purification is achieved without
isopropanol precipitation of purified plasmid DNA,
providing rapid purification as well as a high concentration
of pure plasmid DNA.
The PureYield Plasmid Maxiprep System purifies up to
1mg of plasmid DNA with an A260/A280 >1.7 from 250ml
of overnight bacterial culture, transformed with a
high-copy-number plasmid. The PureYield System
requires a vacuum pump and manifold (e.g., the Vac-Man®
Laboratory Vacuum Manifold, 20-sample [Cat.# A7231]),
a centrifuge with a fixed-angle rotor for lysate clearing and
either a tabletop centrifuge with a swinging bucket rotor
or the Eluator Vacuum Elution Device (Cat.# A1071) for
the final elution step.
Additional Resources for the PureYield Plasmid
Maxiprep System
Technical Bulletins and Manuals
TM280 PureYield Plasmid Maxiprep System
Technical Manual
(www.promega.com
/tbs/tm280/tm280.html)
Promega Publications
eNotes Remove the high-speed spin from
PureYield Plasmid Preps
(www.promega.com
/enotes/applications/ap0101.htm)
Protocols & Applications Guide
www.promega.com
rev. 3/09
D. Wizard® Plus SV Minipreps DNA Purification System
The Wizard® Plus SV Minipreps DNA Purification System
(Cat.# A1330, A1340, A1460, A1470) provides a simple and
reliable method for rapid isolation of plasmid DNA using
a column-based silica membrane (see Figure 9.7 for
overview of method). The entire miniprep procedure can
be completed in 30 minutes or less, depending on the
number of samples processed. The plasmid DNA can be
purified by using either a vacuum manifold like the
Vac-Man® Laboratory Vacuum Manifold (process up to
20 samples) or a microcentrifuge (number of samples
processed depends on rotor size). This system can be used
to isolate any plasmid hosted in E. coli but works most
efficiently when the plasmid is less than 20,000bp in size.
Purified plasmids can be used without further manipulation
for automated fluorescent DNA sequencing as well as for
other standard molecular biology techniques.
Overnight culture
Centrifuge.
Remove culture media.
Resuspend cells.
Lyse cells.
Neutralize.
Clear lysate by
centrifugation.
Insert column in
Collection Tube.
Transfer cleared
lysate and bind DNA.
Wash, removing
solution by
centrifugation.
Elute plasmid DNA.
Figure 9.7. Overview of the Wizard® Plus SV Minipreps DNA
Purification System centrifugation protocol.
The miniprep protocol in Technical Bulletin #TB225 is for
the isolation of plasmid DNA from 1–10ml of overnight
E. coli culture. The yield of plasmid will vary depending
on a number of factors, including the volume of bacterial
culture, plasmid copy number, type of culture medium and
the bacterial strain used as discussed in Factors that Affect
Plasmid DNA Quality and Yield. The DNA binding
capacity of the SV membrane is up to 20µg of high-quality
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PROTOCOLS & APPLICATIONS GUIDE 9-13