Efficient, high-yield isolation of high-quality plasmid DNA
for mammalian cell transfection
Katerina Kourentzi, Craig Branch and Karl H. Hecker
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 • USA
PureLink™ HQ spin column:
a HIGH CAPACITY “mini”
column
Introduction
Traditionally, isolation of plasmid DNA from
bacterial cells relies on alkaline lysis in which
cells (resuspended in buffer containing RNAse A)
are mixed with an alkaline solution containing
detergent, which also denatures the DNA.
Addition of a neutralizing solution, usually
buffered potassium acetate, causes proteins and
genomic DNA to precipitate out of solution,
which allows for easy separation by
centrifugation or filtration. Circular plasmid
DNA simply renatures and remains in solution.
Isopropanol precipitation is commonly used to
recover the plasmid DNA from solution. Silicabased systems have gained in popularity
providing plasmid DNA of adequate purity and
quality for many applications at lower cost and
higher speed compared to anion-exchange
technologies. Plasmid DNA in solution, after the
alkaline lysis, can bind to silica-based resin or
matrix in the presence of high concentrations of
chaotropic salts and be eluted in low salt buffer
or water.
PureLink™ HQ Mini Plasmid DNA Purification
Kit, a silica-based spin column kit, was developed
for isolation of high quality plasmid DNA (high
or low copy number) compatible with restriction
enzyme digestion, PCR, sequencing, bacterial cell
transformation, and mammalian cell transfection.
The kit is based on an alkaline/SDS cell lysis
procedure and selective plasmid DNA binding to
the glass microfiber (GF) filters. Plasmid DNA
bound to the silica-based matrix is subjected to
procedures aimed at removing contaminants
such as genomic DNA and RNA and eluted with
a low salt buffer in a final step.
Plasmid DNA (pcDNA3.1/cat) was purified from
1.5, 3, 6 & 9 x109 E. coli cells. The PureLink™ HQ
protocol was performed for each 1.5 x109 - cell
pellet and batches of clarified cell lysate were
loaded onto the column to achieve the noted cell
numbers. One elution was performed with 100 ml
Elution Buffer.
Typical spin columns for “mini” plasmid DNA
isolation kits perform similarly with a starting
amount of 1.5 x109 E. coli cells. When using
increased amounts of cells, e.g. 9 x109 cells, a
typical mini spin column capacity is far exceeded.
Using the PureLink™ column, it was possible to
isolate  60mg plasmid DNA achieving a 35%
increase in plasmid DNA yields (Figure 1) over a
typical “mini” spin column.
9
Yield (m g) in 100 m l EB
The PureLink™ HQ Mini Plasmid DNA
Purification Kit is specifically designed for
quick
and
efficient
isolation
of
transfection-quality plasmid DNA from a
wide range of bacterial cell numbers with
yields of up to 60 mg of plasmid DNA.
Plasmid DNA isolated is of consistently
high quality as evidenced by its reliable
and reproducible performance in all
relevant downstream applications such as
restriction enzyme digestion, automated
fluorescent sequencing, Gateway® cloning,
transformation of bacterial cells, and most
crucially in mammalian cell transfection.
x 10 cells
1.5
80
70
60
50
40
30
20
10
0
PureLink HQ
3
6
9
Isolation of large plasmids
Using PureLink™ HQ, the plasmids p5L393 (40 kb)
& p1Kbplus (19.5 kb) were isolated from 1 ml
overnight E. coli cultures with yields 3-7 mg/ml of
culture and 260/280 ratios of 1.86  0.07. The
plasmids cut fully with several restriction enzymes
under standard conditions (Figure 2). Genomic
DNA or RNA contamination was not detectable
(< 5%).
2
3 4 5
Kb
40.0
5.0
1.0
The introduction of foreign genes into
mammalian cells by transfection has become an
invaluable tool for the expression and study of
heterologous proteins. A crucial parameter for
successful transfection is the quality of the DNA
(supercoiled to nicked forms ratio, RNA
contamination, endotoxin content). Plasmid DNA
isolated using the PureLink™ HQ Kit provides
consistently high transfection efficiencies with
several mammalian cell lines (Figure 3).
It has to be noted that endotoxin levels of the
plasmid DNA isolated using PureLink™ HQ
were significantly lower than the values reported
in the literature for plasmid preparations using
silica-gel slurries (Qiagen, Product Guide 2003,
pp.119-120). As expected, the endotoxin levels for
the PureLink HQ-isolated DNA (< 100 EU/mg
DNA) were not as low as for the ion exchangeisolated sample DNA. However, all samples were
of transfection quality. As reported in the
literature (Fox, et al., BioTechniques, 2000, 29(3),
pp. 610-619), endotoxin levels greater than 2000
EU/ mg DNA are required to significantly inhibit
transfection for most of the commonly used cell
lines.
Other "mini" kit
Figure 1: Capacity comparison of different
plasmid DNA purification “mini” spin
columns for increased amounts of bacterial
cells (n=3)
1
Mammalian cell transfection
Figure 2: Restriction analysis
of p5L393 and 1Kbplus
isolated with PureLink™ HQ.
Lane 1: p5L393 / Hind III
Lane 2: p5L393 / Ava I
Lane 3: p1Kbplus / BamH I
Lane 4: 1Kb Plus DNA ladder
Lane 5: 1Kb DNA Extension
Ladder
Plasmid DNA isolation from
yeast cells
The two-hybrid system is a widely used method
for studying protein-protein interactions. A
common issue after screening is to confirm
“positive”clones in a re-transformation assay.
The re-transformation assay is based on plasmid
DNA isolation from the positive yeast cells,
transformation of the DNA to E. coli cells,
selection for the “prey” plasmid DNA, and
isolation and analysis of the DNA from several
transformants.
We have successfully used the PureLink HQ
Mini Plasmid DNA Purification Kit to isolate
high quality plasmid DNA from yeast cells.
Digestion with zymolyase, which hydrolyses
poly (b-1,2- glucose) of the yeast cell wall, was
coupled with the PureLink HQ standard
protocol. The total procedure is simple, fast, and
reproducible and can be used routinely to isolate
both low-copy (ARS/CEN-based) and multi-copy
(two micron-based) plasmid DNA from yeast
cells. Isolated plasmid DNA is suitable for PCR
amplification and E. coli transformation (Figure
4).
Kb
Relative b -gal expression
Abstract
PureLink
Anion-exchange mini kit
140
120
100
80
1 2 3 4 5 6 7 8
9
3.0
1.0
MaV203
B1.20
60
40
20
0
GripTite 293 cells
BHK-21cells
Figure 3: Transfection efficiency using plasmid
DNA isolated with PureLink™ HQ or an
anion exchange-based mini kit. 0.3 mg of the
plasmid pcDNA3.1/LacZ/His were transfected
in triplicate into GripTite 293 MSR and BHK21 cells using Lipofectamine 2000.
Expression of b-galactosidase (b-gal) was
measured by hydrolysis of oNPG (48 h post
transfection). Transfection efficiency was
reported as ng b-gal/cm2 culture dish area. In
this figure, relative transfection efficiencies are
expressed as percentages relative to the
efficiency obtained with PureLink HQ mini
kit (=100%) for each cell line (n=3).
Lane 1: 1Kb Plus DNA
ladder
Lane 2-3:
pEXP32/DP1 –pEXP22E2F1
Lane 4-5:
pHybLex/DP1 –pEXP22E2F1
Lane 6-7:
pHybLex/DP1 –pEXP22E2F1
Lane 8-9:
pEXP32/DP1–pEXP22E2F1
Figure 4: Restriction analysis of positive clones
and bait vectors isolated with PureLink HQ
DNA after a two-hybrid screen with the DP-1
dimerization domain as a bait (fragment cloned
into pHybLex/Zeo or pDEST32) in MaV203 or
B1.20 cells. pDEST22 was used for cloning the
“prey” E2F cDNA library. Isolated DNA from
positive cells was transformed to TOP10 cells.
DNA was isolated from the selected
transformants and analyzed by restriction with
Hind III.
Acknowledgements
We would like to thank Dr. Sharon Cates for
valuable help in planning and performing all
transfection experiments, Anna Waters for
providing vectors, and Dr. Phillip Gray for the
two-hybrid positive clones, all from Invitrogen.
Low copy-number plasmid DNA
isolation
Low copy-number binary vectors are
commonly used in Agrobacterium-mediated
plant
transformation.
Prior
to
plant
transformation, A. tumafaciens
cells are
screened for the presence of the vectors.
Minor modifications of the standard
PureLink™ HQ protocol (increased volume of
Neutralization/ Binding buffer, extra wash
with 35% Guanidine HCl- 40% isopropanol,
two 50-ml consecutive elutions) allow fast,
efficient, and reproducible isolation of the lowcopy vector pBI121 (14 kb) from A. tumafaciens
that is of suitable quality for restriction
analysis (Figure 5) and E. coli transformation.
1 2 3
Kb
12.0
5.0
4
5 6
Figure 5: Restriction analysis
of pBI121 isolated from A.
tumafaciens (15 ml culture,
OD600= 1.0-1.5) with
PureLink™ HQ.
Lane 1: 1Kb Plus DNA ladder
Lane 2: pBI121- undigested
Lane 3: pBI121 /Pst I (original purification protocol)
Lane 3-6: pBI121/Pst I (modified purification protocol)
Results and Conclusions
•Quick and efficient isolation of highquality plasmid DNA (low- and highcopy number vectors) from bacterial
and yeast cells
•PureLink™ HQ column has
exceptionally high capacity
•Very low gDNA and RNA
contamination
•Isolation of up to 40 kb plasmid DNA
•Plasmid DNA compatible with all
routine downstream applications such
as restriction enzyme digestion,
transformation of bacterial cells
automated fluorescent sequencing,
Gateway® cloning, in vitro
transcription/translation (data not
shown)
•Low endotoxin content & high
supercoiled to nicked forms ratio
provide consistently high transfection
efficiencies with most of the
commonly used mammalian cell lines.
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 USA • Telephone: 760 603 7200 • FAX: 760 602 6500 • Toll Free Telephone: 800 955 6288 • E-mail: tech_service@invitrogen.com • www.invitrogen.com