Protocols and Applications Guide (US Letter Size) - Promega

||||||||| 9DNA Purification
Luminescence (RLU)
1,000,000
100,000
10,000
1,000
100
Background
293
CHO
3T3
Cos-7
HeLa
Competitor’s Miniprep #1
Competitor’s Miniprep #2
PureYield Minipreps #2
PureYield Minipreps #1
Sample Type
Control Plasmid
No DNA
Figure 9.6. Plasmid DNA prepared using the PureYield Plasmid
Miniprep System consistently works well in transfection
experiments. The pGL4.13[luc2/SV40] Vector (Cat.# E6681) was
prepared using a competing system or the PureYield Plasmid
Miniprep System. Five different commonly used mammalian cell
lines were transfected with the plasmid, and transfection efficiency
was assessed by measuring the luciferase activity using the
ONE-Glo Luciferase Assay System (Cat.# E6110; n = 6).
Additional Resources about the PureYield Plasmid
Miniprep System
Technical Bulletins and Manuals
TB374 PureYield Plasmid Miniprep System
Technical Bulletin
(www.promega.com/tbs/tb374/tb374.html)
Promega Publications
CN021 Transfection-quality plasmid DNA in as
little as ten minutes using the PureYield
Plasmid Miniprep System
(www.promega.com
/cnotes/cn021/cn021_03.htm)
Online Tools
PureYield Plasmid Miniprep System Video Podcast
Protocol
(http://www.promega.com/multimedia/VPA1221/VPA1221.mov)
B. PureYield Plasmid Midiprep System
As research moves from DNA sequencing to analysis of
gene function, the need for rapid methods by which to
isolate large quantities of high-quality plasmid DNA has
increased. The PureYield Plasmid Midiprep System (Cat.#
A2492, A2495) is designed to isolate high-quality plasmid
DNA for use in eukaryotic transfection and in vitro
expression experiments. This midiprep system uses a silica
membrane column to purify plasmid DNA in as little as
Protocols & Applications Guide
www.promega.com
rev. 3/09
7566MF
30 minutes, greatly reducing the time spent on purification
compared to silica resin or other membrane column
methods.
The PureYield Plasmid Midiprep System also
incorporates a unique Endotoxin Removal Wash, designed
to remove substantial amounts of protein, RNA and
endotoxin contaminants from purified plasmid DNA,
improving the robustness of sensitive applications such as
eukaryotic transfection, in vitro transcription and coupled
in vitro transcription/translation. Purification is achieved
without isopropanol precipitation of purified plasmid DNA
or extensive centrifugation, providing rapid purification
as well as a high concentration of pure plasmid DNA from
a single method.
The PureYield Plasmid Midiprep System is designed to
purify 100–200µg of plasmid DNA with an A260/A280 >1.7
from a 50ml overnight culture of bacteria, transformed with
a high-copy-number plasmid, with a total optical density
(O.D.600 of culture × volume of culture) of 100–200. Larger
volumes up to 250ml can be processed, but require greater
volumes of solutions than that supplied with the
PureYield Plasmid Midiprep System.
The PureYield Plasmid Midiprep System is designed for
purification by vacuum using a manifold such as the
Vac-Man® Laboratory Vacuum Manifold (Cat.# A7231),
but there are alternative protocols that use all centrifugation
or vacuum and centrifugation together. All protocols
generate high-quality purified plasmid DNA. A
swinging-bucket tabletop centrifuge or the Eluator
Vacuum Elution Device (Cat.# A1071) is required for the
final elution step regardless of the protocol chosen.
Additional Resources for the PureYield Plasmid
Midiprep System
Technical Bulletins and Manuals
TM253 PureYield Plasmid Midiprep System
Technical Manual
(www.promega.com
/tbs/tm253/tm253.html)
Promega Publications
PN088 Fast, reliable, high-quality midiprep
plasmid purification using the PureYield
Plasmid Midiprep System
(www.promega.com
/pnotes/88/12162_05/12162_05.html)
eNotes Remove the high-speed spin from
PureYield Plasmid Preps
(www.promega.com
/enotes/applications/ap0101.htm)
Citations
Abdel-Latief, M. et al. (2008) An epoxide hydrolase involved
in the biosynthesis of an insect sex attractant and its use to
localize the production site. Proc. Natl. Acad. Sci. USA 105,
8914–9.
PROTOCOLS & APPLICATIONS GUIDE 9-12