Macherey-Nagel NucleoBond Xtra Midi User manual

MACHEREY-NAGELMACHEREY-NAGEL
www.mn-net.com
MN
MACHEREY-NAGEL MACHEREY-NAGEL
MN
Plasmid DNA
Purification
User Manual
NucleoBond®Xtra Midi
NucleoBond®Xtra Maxi
NucleoBond®Xtra Midi Plus
NucleoBond®Xtra Maxi Plus
January 2008/Rev. 04
Procedure in
English
German
French
NEW!
MACHEREY-NAGEL
EN ISO 9001:2000
CERTIFIED
A032494 ManualNBXtraR04en1/7/0/01.08 DP Printed in Germany

Protocol-at-a-glance (Rev. 04)
Plasmid DNA Purification (NucleoBond®Xtra Midi / Maxi)
Midi Maxi
1 Cultivate and harvest
bacterial cells 4,500-6,000 x g
15 min at 4°C
2 Cell lysis high-copy / low-copy high-copy / low-copy
Buffer RES
Buffer LYS
(Important: check Buffer LYS
for precipitated SDS)
8 ml / 16 ml
8 ml / 16 ml
12 ml / 24 ml
12 ml / 24 ml
3 Equilibration of the
column together with
inserted column filter
Buffer EQU
12 ml Buffer EQU
25 ml
4 Neutralization Buffer NEU
8 ml / 16 ml Buffer NEU 12 ml / 24 ml
5 Clarification and
loading of the lysate invert the tube 3 times
load lysate on NucleoBond®Xtra column filter
lysate is simultaneously cleared and loaded
onto the NucleoBond®Xtra column
61
st Washing Buffer EQU
5 ml Buffer EQU 15 ml
7 Discard NucleoBond®
Xtra column filter discard NucleoBond®Xtra
column filter discard NucleoBond®Xtra column filter
82
nd Washing Buffer WASH
8 ml
Buffer WASH
25 ml
9 Elution Buffer ELU
5 ml
Buffer ELU
15 ml
10 Precipitation NucleoBond®Xtra
Midi
NucleoBond®Xtra
Midi Plus
NucleoBond®Xtra
Maxi
NucleoBond®Xtra
Maxi Plus
Isopropanol
3.5 ml
15,000 x g
30 min at 4°C
Isopropanol
3.5 ml
load NucleoBond®
Finalizer
Isopropanol
10.5 ml
15,000 x g
30 min at 4°C
Isopropanol
10.5 ml
load NucleoBond®
Finalizer Large
11 Wash and dry DNA
pellet 70% ethanol
2 ml
15,000 x g
5 min at RT
5-10 min
70% ethanol
2 ml
/
3 x air
70% ethanol
5 ml
15,000 x g
10 min at RT
10-15 min
70% ethanol
5 ml
/
6 x air
12 Reconstitute DNA appropriate
volume of TE buffer
Buffer TRIS
200-800 µl
appropriate
volume of TE buffer
Buffer TRIS
400-1000 µl
MACHEREY-NAGEL GmbH & Co. KG •Neumann-Neander-Str. 6-8 •D-52355 Düren •Germany
Tel.: [phone] •Fax: [phone] •e-mail: [email protected]

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 3
Table of contents
1 Components 5
1.1 Kit contents 5
1.2 Reagents and equipment to be supplied by user 7
2 Kit specifications 8
3 About this User Manual 9
4 NucleoBond®Xtra plasmid purification system 10
4.1 Basic principle 10
4.2 NucleoBond®Xtra anion-exchange columns 10
4.3 Growth of bacterial cultures 12
4.4 Culture volume for high-copy plasmids 13
4.5 Culture volume for low-copy plasmids 14
4.6 Cell lysis 15
4.7 Difficult-to-lyse strains 15
4.8 Setup of NucleoBond®Xtra columns 16
4.9 Filtration and loading of the lysate 17
4.10 Washing of the column 17
4.11 Elution and concentration of plasmid DNA 18
4.12 Determination of DNA yield and quality 21
4.13 Convenient stopping points 21
5 Storage conditions and preparation of working solutions 22
6 Safety instructions - risk and safety phrases 23
7 NucleoBond®Xtra plasmid purification 24
7.1 High-copy plasmid purification (Midi, Maxi) - English 24
7.2 Low-copy plasmid purification (Midi, Maxi) - English 29
7.3 Concentration of NucleoBond®Xtra eluates with the
NucleoBond®Finalizers - English 32
7.4 High-copy plasmid purification (Midi, Maxi) - German 34
7.5 Low-copy plasmid purification (Midi, Maxi) - German 40
7.6 Concentration of NucleoBond®Xtra eluates with the
NucleoBond®Finalizers – German 44

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
4
7.7 High-copy plasmid purification (Midi, Maxi) - French 47
7.8 Low-copy plasmid purification (Midi, Maxi) – French 53
7.9 Concentration of NucleoBond®Xtra eluates with the
NucleoBond®Finalizers – French 57
8 Appendix 60
8.1 Troubleshooting 60
8.2 Ordering information 68
8.3 Product use restriction / warranty 68

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 5
1 Components
1.1 Kit contents
NucleoBond®Xtra
Midi NucleoBond®Xtra Midi Plus
10 preps 50 preps 100 preps 10 preps 50 preps
Cat. No. 740410.10 740410.50 740410.100 740412.10 740412.50
Buffer RES 100 ml 500 ml 1000 ml 100 ml 500 ml
Buffer LYS 4 x 25 ml 500 ml 1000 ml 4 x 25 ml 500 ml
Buffer NEU 100 ml 500 ml 1000 ml 100 ml 500 ml
Buffer EQU 200 ml 2 x 500 ml 2 x 1000 ml 200 ml 2 x 500 ml
Buffer WASH 100 ml 500 ml 1000 ml 100 ml 500 ml
Buffer ELU 60 ml 300 ml 600 ml 60 ml 300 ml
RNase A
(lyophilized) 6 mg 30 mg 2 x 30 mg 6 mg 30 mg
NucleoBond®Xtra
Midi Columns incl.
NucleoBond®Xtra
Midi Column Filters
10 50 100 10 50
NucleoBond®
Finalizers ---1050
30 ml Syringes - - - 10 50
1 ml Syringes - - - 10 50
Buffer TRIS - - - 15 ml 75 ml
Plastic Washers 5 10 10 5 10
User Manual 1 1 1 1 1
For preparation of working solutions and storage conditions see section 5.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
6
1.1 Kit contents continued
NucleoBond®Xtra
Maxi NucleoBond®Xtra Maxi Plus
10 preps 50 preps 100 preps 10 preps 50 preps
Cat. No. 740414.10 740414.50 740414.100 740416.10 740416.50
Buffer RES 150 ml 750 ml 2 x 750 ml 150 ml 750 ml
Buffer LYS 150 ml 750 ml 2 x 750 ml 150 ml 750 ml
Buffer NEU 150 ml 750 ml 2 x 750 ml 150 ml 750 ml
Buffer EQU 500 ml 2 x 1000 ml
500 ml 5 x 1000 ml 500 ml 2 x 1000 ml 500 ml
Buffer WASH 300 ml 1000 ml
500 ml 3 x 1000 ml 300 ml 1000 ml 500 ml
Buffer ELU 180 ml 900 ml 2 x 900 ml 180 ml 900 ml
RNase A
(lyophilized) 10 mg 50 mg 2 x 50 mg 10 mg 50 mg
NucleoBond®Xtra
Maxi Columns incl.
NucleoBond®Xtra
Maxi Column Fil-
ters
10 50 100 10 50
NucleoBond®
Finalizers Large ---1050
30 ml Syringes - - - 10 50
1 ml Syringes - - - 10 50
Buffer TRIS - - - 15 ml 75 ml
Plastic Washers 5 10 10 5 10
User Manual 1 1 1 1 1
For preparation of working solutions and storage conditions see section 5.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 7
1.2 Reagents and equipment to be supplied by user
Chemicals:
•
Isopropanol (room-temperatured)
•
70% ethanol (room-temperatured)
• Buffer for reconstitution of DNA, e.g. TE buffer or sterile H2O (not necessary
for NucleoBondXtra Midi/Maxi Plus kits)
Equipment:
• Standard microbiological equipment for growing and harvesting bacteria (e.g.
inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centri-
fuge with rotor and tubes or bottles for harvesting cells)
• Refrigerated centrifuge capable of reaching 5,000 x gwith rotor for the ap-
propriate centrifuge tubes or bottles (not necessary for NucleoBondXtra
Midi/Maxi Plus kits)
• Centrifugation tubes or vessels with suitable capacity for the volumes specified
in the respective protocol
• NucleoBondXtra Combi Rack (see ordering information) or equivalent holder

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
8
2 Kit specifications
• NucleoBond®Xtra kits are suitable for ultra fast purification of plasmids,
cosmids, and very large constructs (P1 constructs, BACs, PACs) ranging from
3 kbp up to 300 kb. For preparation of working solutions and storage condi-
tions see section 5.
• NucleoBond®Xtra columns are polypropylene columns containing Nu-
cleoBond®Xtra silica resin packed between two inert filter elements. The
columns are available in Midi and Maxi sizes with typical DNA yields of 250 µg
and 1000 µg, respectively.
• All NucleoBond®Xtra columns are resistant to organic solvents such as al-
cohol, chloroform, and phenol and are also suitable for buffers containing de-
naturing agents like formamide, urea, or common detergents like Triton X-100
or NP-40.
• NucleoBond®Xtra silica resin can be used over a wide pH range (pH
2.5–8.5), and can remain in contact with buffers for several hours without any
change in its chromatographic properties.
• The NucleoBond®Xtra column filters are specially designed depth filters
that fit into the NucleoBond®Xtra columns. The filters are inserted ready-to-
use in the NucleoBond®Xtra columns and allow a time-saving simultaneous
clearing of bacterial lysate and loading of cleared lysate onto the Nu-
cleoBond®Xtra column. Furthermore, the use of the column filters avoids the
time-consuming centrifugation step for lysate clearing.
• The NucleoBond®Xtra column filters allow complete removal of precipitate
even with large lysate volumes without clogging and avoid shearing of large
DNA constructs, such as PACs or BACs by the gentle depth filter effect.
• The NucleoBond®Xtra Midi Plus and NucleoBond®Xtra Maxi Plus kits ad-
ditionally contain the NucleoBond®Finalizers and NucleoBond®Finalizers
Large, respectively. These tools for a fast concentration and desalination of
eluates are suitable for most plasmids and cosmids ranging from 2-50 kbp with
recovery efficiencies from 40-90% (depending on elution volume).
•
NucleoBond®Finalizer is a polypropylene syringe filter containing a special
silica membrane. The NucleoBond®Finalizer provides a binding capacity of
500 µg, whereas the NucleoBond®Finalizer Large can hold up to 2000 µg
plasmid DNA.
• Due to the small dead volumes of the NucleoBond®Finalizers the plasmid
DNA can be eluted with a concentration up to 3 µg/µl (see section 4.11, Table
4 and 5 for dependence of concentration on elution volume).
• All NucleoBond®Finalizers are resistant to organic solvents such as alcohol,
chloroform, and phenol and are free of endotoxins.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 9
3 About this User Manual
The following section 4 provides you with a detailed description of the NucleoBond®
Xtra purification system and important information about cell growth, cell lysis, and
the subsequent purification steps. Sections 5 and 6 inform you about storage, buffer
preparation, and safety instructions.
First-time users are strongly advised to read these chapters thoroughly before using
this kit. Experienced users can directly proceed with the purification protocols (sec-
tion 7) or just use the Protocol-at-a-glance for a quick reference.
Each procedural step in the purification protocol is arranged like the following exam-
ple taken from section 7.1:
Midi Maxi
5 Cell lysis
Check Lysis Buffer LYS for precipitated SDS prior to use. If a white precipitate is
visible, warm the buffer for several minutes at 30-40°C until precipitate is dis-
solved completely. Cool buffer down to room temperature (20-25°C).
Add Lysis Buffer LYS to the suspension.
Mix gently by inverting the tube 5 times. Do not vortex as this will shear and
release contaminating chromosomal DNA from cellular debris into the suspension.
Incubate the mixture at room temperature (20-25°C) for 5 min.
Note: Increase LYS buffer volume proportionally if more than the recommended cell mass
is used (see section 4.6 for information on optimal cell lysis).
8 ml 12 ml
If you are performing a Midi prep to purify plasmid DNA you will find volumes or incu-
bation times in the white boxes. For Maxi preps please refer to the black boxes.
The name of the buffer, buffer volume, incubation times, repeats or important han-
dling steps are emphasized in bold type within the instruction. Additional notes or
optional steps are printed in italic. The exclamation point marks information and hints
that are essential for a successful preparation.
In the example shown above you are asked to check the Lysis Buffer LYS prior to
use and then to lyse the resuspended cell pellet in 8 ml of Buffer LYS when per-
forming a Midi prep and in 12 ml for a Maxi prep. Follow the handling instructions
exactly and note the given hints for protocol alterations.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
10
4 NucleoBond®Xtra plasmid purification system
4.1 Basic principle
The bacterial cells are lysed by an optimized set of newly formulated buffers based
on the NaOH/SDS lysis method of Birnboim and Doly.
After equilibration of the NucleoBond®Xtra column together with the corresponding
NucleoBond®Xtra column filter, the entire lysate is loaded by gravity flow and si-
multaneously cleared by the specially designed column filter.
Plasmid DNA is bound to the NucleoBond®Xtra silica resin.
After an efficient washing step the plasmid DNA is eluted, precipitated, and easily
dissolved in any suitable buffer (e.g. low-salt buffer or water) for further use.
4.2 NucleoBond®Xtra anion-exchange columns
NucleoBond®Xtra is a silica-based anion-exchange resin, developed by
MACHEREY-NAGEL and covered under European Patent EP 0 496 822. It is devel-
oped for routine separation of different classes of nucleic acids like oligonucleotides,
RNA, and plasmids.
NucleoBond® Xtra silica resin consists of hydrophilic, macroporous silica beads
functionalized with MAE (methyl-amino-ethanol). The dense coating of this functional
group provides a high overall positive charge density under acidic pH conditions that
permits the negatively charged phosphate backbone of plasmid DNA to bind with
high specificity (Figure 1).
Figure 1 Ionic interaction of the positively charged methyl-hydroxyethyl-amino group with the
negative phosphate oxygen of the DNA backbone. In contrast to the widely used DEAE
(diethylaminoethanol) group, the hydroxy group of methyl-hydroxyethyl-amin can be in-
volved in additional hydrogen bonding interactions with the DNA.
Birnboim, H. C. and Doly, J., (1979) Nucl. Acids Res. 7, 1513-1523

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 11
Due to a specialized manufacturing process that is strictly controlled and monitored,
the NucleoBond®Xtra silica beads are uniform in diameter and contain particularly
large pores. These special properties allow optimized flow rates and sharp, well-
defined elution profiles. NucleoBond®Xtra can separate distinct nucleic acid species
from each other and from proteins, carbohydrates, and other unwanted cellular com-
ponents over an exceptionally broad range of salt concentrations (Figure 2).
All contaminants from proteins to RNA are washed from the column, the positive
charge of the resin is neutralized by a pH shift to slightly alkaline conditions, and pure
plasmid DNA is eluted in a high-salt elution buffer.
The purified nucleic acid products are suitable for use in the most demanding mo-
lecular biology applications, including transfection, in vitro transcription, automated or
manual sequencing, cloning, hybridization, and PCR.
Figure 2 Elution profile of NucleoBond®Xtra silica resin at pH 7.0
The more interactions a nucleic acid can form between the phosphate backbone and the
positively charged resin the later it is eluted with increasing salt concentration. Large nu-
cleic acids carry more charges than short ones, double stranded DNA more than single
stranded RNA.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
12
4.3 Growth of bacterial cultures
Yield and quality of plasmid DNA highly depend on the type of culture media and
antibiotics, the bacterial host strain, the plasmid type, size, and copy number.
For standard high-copy plasmids LB (Luria-Bertani) medium is recommended. The
cell culture should be incubated at 37°C with constant shaking (200-250 rpm) pref-
erably 12-16 h over night. Use flasks of at least three or four times the volume of the
culture volume to provide a growth medium saturated with oxygen. Alternatively, rich
media like 2xYT (Yeast/Tryptone), TB (Terrific Broth) or CircleGrow can be used. In
this case bacteria grow faster, reach the stationary phase much earlier than in LB
medium (12 h), and higher cell masses can be reached. However, this does not
necessarily yield more plasmid DNA. Overgrowing a culture might lead to a higher
percentage of dead or starving cells and the resulting plasmid DNA might be partially
degraded or contaminated with chromosomal DNA. To find the optimal culture condi-
tions, the culture medium and incubation times have to be optimized for each host
strain/plasmid construct combination individually.
Cell cultures should be grown under antibiotic selection at all times to ensure plas-
mid propagation. Without this selective pressure, cells tend to lose a plasmid during
cell division. Since bacteria grow much faster without the burden of a high-copy
plasmid, they take over the culture rapidly and the plasmid yield goes down regard-
less of the cell mass. Table 1 gives information on concentrations of commonly used
antibiotics.
Table 1: Information about antibiotics according to Maniatis
Antibiotic Stock solution
(concentration) Storage Working concentration
Ampicillin 50 mg/ml in H2O -20°C 50-100 µg/ml
Chloramphenicol 34 mg/ml in EtOH -20°C 25-170 µg/ml
Kanamycin 10 mg/ml in H2O -20°C 10-50 µg/ml
Streptomycin 10 mg/ml in H2O -20°C 10-50 µg/ml
Tetracycline 5 mg/ml in EtOH -20°C 10-50 µg/ml
The E. coli host strain mostly influences the quality of the plasmid DNA. Whereas
strains like DH5or XL1-Blue usually produce high quality super-coiled plasmid
DNA, other strains like e.g. HB101 with high levels of endonuclease activity might
yield lower quality plasmid giving poor results in downstream applications like enzy-
matic restriction or sequencing.
Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor,
Cold Spring, New York 1982.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 13
The type of plasmid, especially the size and the origin of replication (ori) has a
crucial influence on DNA yield. In general, the larger the plasmid or the cloned insert
is, the lower is the expected DNA yield due to a lower copy number. Even a high-
copy construct based on a ColE1 ori can behave like a low-copy vector in case of a
large or unfavorable insert. In addition, the ori itself influences the yield by factor
10-100. Thus plasmids based on e.g. pBR322 or pACYC, cosmids or BACs are
maintained at copy numbers < 20 down to even only 1, whereas vectors based on
e.g. pUC, pBluescript or pGEM can be present in several hundred copies per cell.
Therefore, all the above mentioned factors should be taken into consideration if a
particular DNA yield has to be achieved. Culture volume and lysis procedures have to
be adjusted accordingly.
4.4 Culture volume for high-copy plasmids
Due to the influence of growth media (TB, CircleGrow, 2xYT), growth conditions
(shaking, temperature), host strain or type of plasmid insert etc. the final amount of
cells in a bacterial culture can vary over a wide range. By rule of thumb, 1 liter of E.
coli culture with an OD600 of 1 consists of 1 x 1012 cells and yields about 1.5-1.8 g cell
wet weight. Overnight cultures grown in LB medium usually reach an OD600 of 3-6
under vigorous shaking in flasks. Fermentation cultures even reach an OD600 of 10
and more. The expected DNA yield for a high-copy plasmid is approximately 1 mg
per gram cell wet weight.
It is therefore important to adjust the cell mass rather than the culture volume for the
best plasmid purification results. But since the cell mass or cell wet weight is tedious
to determine it was replaced in this manual by the mathematical product of optical
density at 600 nm (OD) and culture volume (V) - two variables that are much easier
to measure.
ODV = OD600 x Vol [ml]
Note that the culture samples should be diluted if OD600 exceeds 0.5 in order to in-
crease proportionally with cell mass. For a well grown E. coli culture a 1:10 dilution
with fresh culture medium is recommended. The measured OD600 is then multiplied
with the dilution factor 10 to result in a theoretical OD600 value. This OD600 is used in
Table 2 to determine the appropriate culture volume.
Table 2: Recommended culture volumes for high-copy plasmids
Recommended culture volume for
NucleoBond®
Xtra kit
Pellet
wet
weight
Rec.
ODV OD600 =
2
OD600 =
4
OD600 =
6
OD600 =
8
OD600 =
10
Midi 0.75 g 400 200 ml 100 ml 66 ml 50 ml 40 ml
Maxi 2.25 g 1200 600 ml 300 ml 200 ml 150 ml 120 ml
Table 2 shows recommended ODVs and the corresponding pairs of OD600 and cul-
ture volume that can be easily handled using the standard kit protocol lysis buffer
volumes.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
14
4.5 Culture volume for low-copy plasmids
NucleoBond®Xtra kits are designed for isolation of high-copy plasmids (up to sev-
eral hundred copies/cell) as well as low-copy plasmids (<20 copies/cell). However,
when purifying low-copy plasmids, the cell mass and the lysis buffer volumes should
be increased at least by factor 2 to provide enough DNA to utilize the columns´ bind-
ing capacity. Table 3 shows recommended ODVs and the corresponding pairs of
OD600 and culture volume for low-copy plasmid cell cultures (for detailed information
on calculating ODV = OD600 x Vol. refer to section 4.4).
For higher yields, it is advantageous to increase the cell culture and lysis buffer vol-
umes even more, e.g. by factor 3-5. In this case additional lysis buffer can be ordered
separately (see section 8.2 for ordering information). Furthermore, a centrifuge
should be used for lysate clarification instead of the provided NucleoBond®Xtra
column filters since their capacity for precipitate is limited.
Table 3: Suggested culture volumes for low-copy plasmids
Recommended culture volume for
NucleoBond®
Xtra kit
Pellet
wet
weight
Rec.
ODV OD600 =
2
OD600 =
4
OD600 =
6
OD600 =
8
OD600 =
10
Midi 1.50 g 800 400 ml 200 ml 133 ml 100 ml 80 ml
Maxi 4.50 g 2400 1200 ml 600 ml 400 ml 300 ml 240 ml

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 15
4.6 Cell lysis
The bacterial cell pellet is resuspended in Buffer RES and lysed by a sodium hy-
droxide/SDS treatment with Buffer LYS. Proteins, as well as chromosomal and plas-
mid DNA are denatured under these conditions. RNA is degraded by DNase-free
RNase A. Neutralization Buffer NEU, containing potassium acetate, is then added to
the lysate, causing SDS to precipitate as KDS (potassium dodecyl sulfate) and pull-
ing down proteins, chromosomal DNA, and other cellular debris. The potassium
acetate buffer also neutralizes the lysate. Plasmid DNA can revert to its native super-
coiled structure and remains in solution.
The NucleoBond®Xtra buffer volumes (standard protocol) are adjusted to ensure
optimal lysis for culture volumes, appropriate for high-copy plasmids according to
section 4.4, Table 2. Using too much cell material leads to inefficient cell lysis and
precipitation and might reduce plasmid yield and purity. Therefore, lysis buffer vol-
umes should be increased when applying larger culture volumes in case of e.g. low-
copy vector purification (section 4.5, Table 3).
By rule of thumb, calculate the necessary lysis buffer volumes for RES, LYS, and
NEU as follows:
Vol. [ml] = Culture Volume [ml] x OD600 / 50
For example, if 200 ml of a low-copy bacterial culture (OD600 = 4) is to be lysed, the
appropriate volumes of lysis buffers RES, LYS, and NEU are 16 ml each. If more ly-
sis buffer is needed than is provided with the kit, an additional buffer set including
buffers RES, LYS, NEU, and RNase A can be ordered separately. Please refer to
section 8.2 for ordering information.
By using sufficient amounts of lysis buffer, lysis time can be limited to 3-4 minutes
and should not exceed 5 minutes. Prolonged exposure to alkaline conditions can ir-
reversibly denature and degrade plasmid DNA and liberate contaminating chromo-
somal DNA into the lysate.
4.7 Difficult-to-lyse strains
For plasmid purification of e.g. Gram-positive bacteria or strains with a more resistant
cell wall it might be advantageous to start the preparation with a lysozyme treatment.
Therefore, resuspend the cell pellet in Buffer RES containing 2 mg/ml lysozyme and
incubate at 37°C for 30 minutes. Proceed then with the lysis procedure according to
the NucleoBond®Xtra standard protocol.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
16
4.8 Setup of NucleoBond®Xtra columns
Ideally the NucleoBond®Xtra Midi or Maxi Columns are placed into a Nu-
cleoBond®Xtra Combi Rack (see section 8.2 for ordering information). They are
held either by the collar ring of the cartridges or by the Plastic Washers included in
the kit to individually adjust the height of each column (see Figure 3). The Plastic
Washers can also be used to hold the columns on top of suitable collecting tubes or
flasks. The NucleoBond®Xtra Combi Rack can be used in combination with Nu-
cleoBond®PC 100, 500, and 2000 as well. Note that the NucleoBond®Xtra Midi
Columns can also be placed in the NucleoBond®Rack Large (Cat. No. 740563).
AB
Figure 3
Setup of NucleoBond®Xtra Midi/Maxi Columns with the NucleoBond®Xtra Combi Rack
A: Setup for clarification, loading, and first washing step, B: Setup for elution.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 17
4.9 Filtration and loading of the lysate
After the alkaline lysis, the sample has to be cleared from cell debris and precipitate
to ensure high plasmid purity and a fast column flow rate. This is achieved by passing
the solution through a NucleoBond®Xtra column filter which is provided already
inserted into the NucleoBond®Xtra column.
Midi Maxi
The NucleoBond®Xtra column filters are designed to eliminate the centrifugation
step after alkaline lysis. They are pre-wet during column equilibration and allow a
time-saving simultaneous clearing of bacterial lysate and loading of the Nu-
cleoBond®Xtra column.
Compared to lysate clearing by centrifugation or syringe filters the NucleoBond®
Xtra column filter furthermore avoids shearing of large DNA constructs such as
PACs or BACs by the gentle depth filter effect (filtration occurs on the surface of the
filter as well as inside the filter matrix). Its special material and design lead to very
rapid passage of the lysate through the filter and even very large lysate volumes can
be applied without the risk of clogging. This is especially important for e.g. low-copy
plasmid purification. However, if more than the recommended cell mass (see section
4.4, Table 2, section 4.5, Table 3) was lysed, it might be advantageous to use a cen-
trifuge for lysate clarification rather than the provided column filters due to their lim-
ited precipitate capacity.
4.10 Washing of the column
The high salt concentration of the lysate prevents proteins and RNA from binding to
the NucleoBond®Xtra column (see section 4.2, Figure 2). However, to remove all
traces of contaminants and to purge the dead volume of the NucleoBond®Xtra col-
umn filters it is important to wash the column and the filter in two subsequent wash-
ing steps.
First apply Buffer EQU to the funnel rim of the filter to wash all residual lysate out of
the filter onto the column. Do not just pour the buffer inside the filter. Then pull out
and discard the column filter or remove the filter by turning the column upside down.
It is essential to wash the NucleoBond®Xtra column without filter for a second time
with Wash Buffer WASH. This ensures highest yields with best achievable purity.
NucleoBond®Xtra
column filter
NucleoBond®Xtra
column

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
18
4.11 Elution and concentration of plasmid DNA
Elution is carried out under high-salt conditions and by a shift of pH from 6.5 to 9.0.
Under these alkaline conditions the positive charge of the anion-exchange resin is
neutralized and plasmid DNA is released. For any downstream applications it is nec-
essary to precipitate the DNA and to remove salt and all traces of alcohol since they
disturb or inhibit enzymatic activity needed for restriction or sequencing reactions.
All NucleoBond®Xtra eluates already contain enough salt for an isopropanol pre-
cipitation of DNA. Therefore the precipitation is started by directly adding 0.7 volumes
of isopropanol. To prevent co-precipitation of salt, use room-temperature (20-25°C)
isopropanol only and do not let the plasmid DNA solution drop into a vial with iso-
propanol but add isopropanol to the final eluate and mix immediately.
Afterwards either follow the centrifugation protocol given after the NucleoBond®Xtra
purification protocol or follow the support protocol for the NucleoBond®Finalizers in
section 7.3 to eliminate the time-consuming centrifugation steps for precipitation (use
of NucleoBond®Finalizers is only recommended for vector sizes smaller than
50 kbp).
The NucleoBond®Finalizers are designed for quick concentration and desalination
of plasmid and cosmid DNA eluates that are obtained by anion-exchange chromatog-
raphy based DNA purification. The sample is precipitated with isopropanol as men-
tioned above and loaded onto a special silica membrane by means of a syringe. After
an ethanolic washing step the membrane is dried by pressing air through the filter.
Elution of pure DNA is carried out with slightly alkaline low salt buffers like Buffer
TRIS (5 mM Tris/HCl, pH 8.5, provided with all NucleoBond®Xtra Plus kits) or TE
buffer (10 mM Tris/HCl, pH 7.5, 1 mM EDTA).
For maximum yield it is recommended to perform the elution step twice. The
first elution step is done using fresh buffer whereas in the second elution step the
eluate from the first elution is reapplied on the NucleoBond®Finalizer to allow com-
plete solubilization of the plasmid.
DNA recovery highly depends on the used elution buffer volume. Large volumes
result in a high recovery of up to 90% but in a lower DNA concentration. Small elution
volumes on the other hand increase the concentration but at the cost of DNA yield.
If a small volume is chosen, make sure to recover as much eluate as possible from
the syringe and NucleoBond®Finalizer by pressing air through the NucleoBond®
Finalizer several times after elution and collecting every single droplet to minimize
the dead volume.
Figure 4 and 5 illustrate exemplarily how DNA recovery and final DNA concentration
depend on the buffer volume which is used for elution of DNA from the NucleoBond®
Finalizer and NucleoBond®Finalizer Large, respectively.

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04 19
0
10
20
30
40
50
60
70
80
90
100
0 200 400 600 800 1000
Elution Volume [l]
Recovery [%]
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
Concentration [g/l]
Recovery
Concentration
Figure 4 Final DNA recovery and concentration after NucleoBond®Finalizer application
A NucleoBond®Xtra Midi eluate containing 250 µg plasmid DNA (8 kbp) was loaded onto a
NucleoBond®Finalizer and eluted two-fold with increasing volumes of TE buffer.
The NucleoBond®Finalizer is designed to hold a maximum of 500 µg DNA and is
therefore ideally suited to be used in combination with NucleoBond®Xtra Midi.
Maximum DNA recovery can be achieved by using > 600 µl of elution buffer. For a
higher concentration experienced users can lower the elution buffer volume to 400-
200 µl.
Table 4 gives an overview about recovery and concentration of different amounts of
plasmid DNA loaded onto a NucleoBond®Finalizer. DNA was eluted two-fold with
increasing volumes of TE-EF. Please refer to this tables to select an elution buffer
volume that meets your needs best.
Table 4: DNA recovery and concentration for the NucleoBond®Finalizer
Elution Volume
100 µl 200 µl 400 µl 600 µl 800 µl 1000 µl
35 % 60 % 70 % 75 % 75 % 75 %
500 µg
2.5 µg/µl 2.3 µg/µl 1.2 µg/µl 0.8 µg/µl 0.6 µg/µl 0.5 µg/µl
40 % 65 % 75 % 80 % 80 % 80 %
250 µg
1.9 µg/µl 1.1 µg/µl 0. 6 µg/µl 0.4 µg/µl 0.3 µg/µl 0.2 µg/µl
45 % 70 % 80 % 85 % 85 % 85 %
100 µg
0.7 µg/µl 0.4 µg/µl 0.2 µg/µl 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl
30 % 75 % 85 % 90 % 90 % 90 %
Loaded DNA
50 µg
0.3 µg/µl 0.2 µg/µl 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl <0.1 µg/µl
DNA recovery
DNA concentration

Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
20
0
10
20
30
40
50
60
70
80
90
100
0 200 400 600 800 1000
Elution Volume [l]
Recovery [%]
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Concentration [g/l]
Recovery
Concentration
Figure 5 Final DNA recovery and concentration after NucleoBond®Finalizer Large application
A NucleoBond®Xtra Maxi eluate containing 1000 µg plasmid DNA (8 kbp) was loaded onto
a NucleoBond®Finalizer Large and eluted two-fold with increasing volumes of TE buffer.
NucleoBond®Xtra Maxi eluates are easily concentrated with a NucleoBond®Fi-
nalizer Large which is able to bind up to 2000 µg plasmid DNA. Maximum DNA re-
covery can be achieved by using > 800 µl of elution buffer. For a higher concentration
experienced users can lower the elution buffer volume to 600-400 µl.
Table 5 gives an overview about recovery and concentration of different amounts of
plasmid DNA loaded onto a NucleoBond®Finalizer Large. DNA was eluted two-fold
with increasing volumes of TE-EF. Please refer to this tables to select an elution
buffer volume that meets your needs best.
Table 5: DNA recovery and concentration for the NucleoBond®Finalizer Large
Elution Volume
100 µl 200 µl 400 µl 600 µl 800 µl 1000 µl
5 % 30 % 65 % 80 % 85 % 90 %
1500 µg
1.9 µg/µl 3.2 µg/µl 2.9 µg/µl 2.2 µg/µl 1.7 µg/µl 1.4 µg/µl
5 % 35 % 70 % 85 % 90 % 90 %
1000 µg
1.3 µg/µl 2.5 µg/µl 2.1 µg/µl 1.6 µg/µl 1.2 µg/µl 1.0 µg/µl
10 % 40 % 70 % 85 % 90 % 90 %
500 µg
1.3 µg/µl 1.4 µg/µl 1.0 µg/µl 0.8 µg/µl 0.6 µg/µl 0.5 µg/µl
15 % 45 % 70 % 80 % 85 % 90 %
Loaded DNA
100 µg
0.4 µg/µl 0.3 µg/µl 0.2 µg/µl 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl
DNA recovery
DNA concentration