Protocols and Applications Guide (US Letter Size) - Promega

||||||||| 9DNA Purification
depending on the number of samples processed and the
protocol used. The purified DNA can be used for automated
fluorescent DNA sequencing, cloning, labeling, restriction
enzyme digestion or in vitro transcription/translation
without further manipulation.
Table 9.9. Percent Recovery Versus Double-Stranded
DNA Fragment Size Using the Wizard® SV Gel and PCR
Clean-Up System.
DNA Fragment Size
Percent Recovery
55bp
26%
70bp
39%
85bp
55%
100bp
84%
500bp
89%
1,000bp
92%
3,199bp
95%
9,416bp
95%
23,130bp
47%
Table 9.10. Effect of Various PCR Additives on Percent
Recovery of a 1,000bp PCR Product Using the Direct
Purification Method and the Wizard® SV Gel and PCR
Clean-Up System.
PCR Additive
no additive
1M betaine
1M Q-Solution
0.1% Triton® X-100
0.1% Tween®-20
0.1% NP-40
5% glycerol
5% formamide
5% DMSO
0.5M tetramethylene sulfoxide
0.4M sulfolane
0.4M 2-pyrollidone
1mM tartrazine
1% Ficoll®-400
Percent Recovery1 100%
94%
97%
92%
87%
82%
87%
90%
87%
94%
94%
95%
100%
100%
1 Percent recovery shown is relative to the “no additive” recovery.
For direct purification from a reaction, note that any nucleic
acid present in solution will be isolated. Therefore, if an
amplification reaction has more than one product, all
fragments will be present in the eluted DNA. If you are
interested in isolating a single amplimer, separate the
reaction products on an agarose gel and cut out the band
desired prior to purification.
When purifying DNA from an agarose slice, the primary
consideration is to melt the agarose so the DNA is available
for binding to the silica membrane. The purified DNA can
then be used for cloning or sequencing.
Protocols & Applications Guide
www.promega.com
rev. 3/09
Additional Resources for the Wizard® SV Gel and PCR
Clean-Up System
Technical Bulletins and Manuals
TB308 Wizard® SV Gel and PCR Clean-Up System
Technical Bulletin
(www.promega.com/tbs/tb308/tb308.html)
Promega Publications
PN082 Wizard® SV Gel and PCR Clean-Up
System
(www.promega.com
/pnotes/82/10203_02/10203_02.html)
eNotes Removal of ethidium bromide and calf
intestinal alkaline phosphatase using the
Wizard® SV Gel and PCR Clean-Up
System
(www.promega.com
/enotes/applications/ap0045_tabs.htm)
Citations
Suzuki, A. et al. (2006) NDR2 acts as the upstream kinase
of ARK5 during insulin-like growth factor-1 signaling.
J. Biol. Chem. 281, 13915–21.
A deletion mutation of the serine/threonine protein kinase
NDR2 was created by PCR using two mutagenesis primers
and two plasmid-based primers of the clone. After
amplification, the two products were run on a 1% agarose
gel and extracted using the Wizard® SV Gel and PCR
Clean-Up System. The purified fragments were mixed,
annealed, reamplified and then digested prior to cloning
into an expression vector. The human colorectal cancer cell
lines HCT-116, DLD-1, and SW480 used in the study were
seeded into a 24-well plate at 5 × 104 cells/well and
transfected using the TransFast Transfection Reagent.
The transfection was assessed with a green fluorescent
protein expression vector.
PubMed Number: 16488889
B. Wizard® SV 96 PCR Clean-Up System
To purify 96 amplification reactions at once, use the
Wizard® SV 96 PCR Clean-Up System (Cat.# A9340, A9341,
A9342, A9345) with a 96-well vacuum manifold (Vac-Man®
96 Vacuum Manifold) and a vacuum pump capable of
generating 15–20 inches of mercury or the equivalent. This
system is designed to purify 100bp to 10kb PCR products
directly from a reaction with typical recovery >90% as seen
in Figure 9.18.
PROTOCOLS & APPLICATIONS GUIDE 9-32