Macherey-Nagel NucleoSpin 740727.10 User manual

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Plasmid DNA
purication
User manual
NucleoSpin®Plasmid EasyPure
August 2013 / Rev. 01
A0xxxxx /01308

Plasmid DNA purication
Protocol-at-a-glance (Rev. 01)
MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Germany
Tel.: [phone] · Fax: [phone] · [email protected] · www.mn-net.com
NucleoSpin®
Plasmid EasyPure
1Cultivate
and harvest bacterial
cells
12,000 x g,
30 s
2Cell lysis 150 μL Buffer A1
250 μL Buffer A2
RT, up to 2 min
350 μL Buffer A3
3Clarication of the lysate
> 12,000 x g,
3 min
4Bind DNA Load supernatant
1,000–2,000 x g,
30 s
5Wash and dry silica
membrane 450 μL Buffer AQ
> 12,000 x g,
1 min
6 Elute DNA 50 μL Buffer AE
RT, 1 min
> 12,000 x g,
1 min

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Plasmid DNA purication
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Table of contents
1 Components 4
1.1 Kit contents 4
1.2 Reagents, consumables, and equipment to be supplied by user 5
1.3 About this user manual 5
2 Product description 6
2.1 Basic principle 6
2.2 Kitspecications 6
2.3 Growth of bacterial cultures 7
2.4 Elution procedures 8
3 Storage conditions and preparation of working solutions 10
4 Safety instructions 11
4.1 Risk and safety phrases 11
4.2 GHSclassication 12
5 NucleoSpin®Plasmid EasyPure protocol 14
6 Appendix 16
6.1 Troubleshooting 16
6.2 Ordering information 19
6.3 References 19
6.4 Product use restriction / warranty 20

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1 Components
1.1 Kit contents
NucleoSpin®Plasmid EasyPure
10 preps 50 preps 250 preps
REF 740727.10 740727.50 740727.250
Resuspension Buffer A1 5 mL 15 mL 75 mL
Lysis Buffer A2 5 mL 15 mL 75 mL
Neutralization Buffer A3 5 mL 20 mL 100 mL
Wash Buffer AQ
(Concentrate)* 6 mL 6 mL 30 mL
Elution Buffer AE** 15 mL 15 mL 30 mL
Liquid RNase A 2 mg 6 mg 30 mg
NucleoSpin®Plasmid
EasyPure Columns
(dark blue rings)
10 50 250
Collection Tubes (2 mL) 10 50 250
Short protocol 111
* For preparation of working solutions and storage conditions see section 3.
**Composition of Elution Buffer AE: 5 mM Tris/HCl, pH 8.5

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1.2 Reagents, consumables, and equipment to be supplied
by user
Reagents
• 96–100 % ethanol
Consumables
• 1.5 mL microcentrifuge tubes for sample lysis and DNA elution
• Disposable pipette tips
Equipment
• Manual pipettors
• Centrifuge for microcentrifuge tubes
• Vortex mixer
• Personal protection equipment (lab coat, gloves, goggles)
1.3 About this user manual
It is strongly recommended reading the detailed protocol sections of this user manual
if the NucleoSpin® Plasmid EasyPure kitisusedforthersttime. Experienced users,
however, may refer to the Protocol-at-a-glance instead. The Protocol-at-a-glance is
designed to be used only as a supplemental tool for quick referencing while performing
thepuricationprocedure.
All technical literature is available on the internet at www.mn-net.com. Please visit
the MACHEREY-NAGEL website to verify that you are using the latest revision of this
user manual.
Please contact Technical Service regarding information about changes of the current
user manual compared to previous revisions.

Plasmid DNA purication
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2 Product description
2.1 Basic principle
With the NucleoSpin® Plasmid EasyPure method, the pelleted bacteria are
resuspended (Buffer A1) and plasmid DNA is liberated from the E. coli host cells by
SDS / alkaline lysis (Buffer A2). Buffer A3 neutralizes the resulting lysate and creates
appropriate conditions for binding of plasmid DNA to the silica membrane of the
NucleoSpin®Plasmid EasyPure Column. Precipitated protein, genomic DNA, and
cell debris are then pelleted by a centrifugation step. The supernatant is loaded onto a
NucleoSpin® Plasmid EasyPure Column.
With the NucleoSpin®Plasmid EasyPure kit contaminations like salts, metabolites,
nucleases, and soluble macromolecular cellular components are removed by only a
singlewashingstepwithBufferAQ.PureplasmidDNAisnallyelutedunderlowionic
strength conditions with slightly alkaline Buffer AE (5 mM Tris/HCl, pH 8.5).
2.2 Kitspecications
• The NucleoSpin®Plasmid EasyPure kits are designed for the rapid, small-
scale preparation of highly pure plasmid DNA (mini preps).
• The NucleoSpin®Plasmid EasyPure Column features a new specially
treated silica membrane which allows speeding up the procedure by a combined
washing and drying step. No additional steps are necessary if nuclease rich
host strains are used. The number of washing and drying steps is reduced from
3 to only 1!
• LyseControl: The Lysis Buffer A2 contains a blue pH indicator to ensure
complete neutralization for maximum yield.
• ThepuriedplasmidDNAissuitableforapplicationslikeautomateduorescent
DNA sequencing, PCR, or any kind of enzymatic manipulation.
* For preparation of working solutions and storage conditions see section 3.
**Composition of Elution Buffer AE: 5 mM Tris/HCl, pH 8.5

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Table1:Kitspecicationsataglance
Parameter NucleoSpin®Plasmid EasyPure
Culture volume 2–10 mL
Typical yield 15–30μg
Elution volume 50μL
Binding capacity 35μg
Vectors < 15 kbp
Preparation time* 14min /6preps
Format Mini spin column
2.3 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, or copy number.
For cultivation of bacterial cells harbouring standard high-copy plasmids, we
recommend LB (Luria Bertani) medium. The cell culture should be incubated at
37 °C with constant shaking (200–250 rpm) preferably 12–16 h over night. Usually an
OD of 3–6canbeachieved.Alternatively,richmedialike2 xYT(Yeast / Tryptone),TB
(Terric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
ndtheoptimalcultureconditions,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 plasmid
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 regardless of the
cell mass. Table 2 gives information on concentrations of commonly used antibiotics.
* Hands-on-time

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Table 2: Information about antibiotics according to Maniatis*
Antibiotic Stock solution
(concentration)
Storage Working
concentration
Ampicillin 50 mg/mL in H2O-20 °C 20–50μg/mL
Carbenicillin 50 mg/mL in H2O-20 °C 20–60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
As rule of thumb use 5 mLofawellgrownLBcultureasgiveninthekitspecications.
However, the culture volume can be increased if the cell culture grows very poorly or
has to be decreased if, e.g., very rich culture media were used. Refer to Table 3 to
choose the best culture volume according to the optical density at 600 nm (OD600).
Table 3: Recommended culture volumes for NucleoSpin®Plasmid EasyPure
OD600 123456
Culture volume 15 mL 8 mL 5 mL 4 mL 3 mL 2 mL
Note, if too much bacterial material is used, the lysis and precipitation steps become
inefcientcausingdecreasedyieldandplasmidquality!Ifmorethantherecommended
amount of cells shall be processed increase all lysis buffers proportionally.
2.4 Elution procedures
The elution buffer volume and method can be adapted to the subsequent downstream
application to achieve higher yield and / or concentration than the standard method
(recovery about 70–90 %):
• Higher yield in general, especially for larger constructs: Heat elution buffer
to 70°C, add 50–100μL to the NucleoSpin®Plasmid EasyPure Column and
incubate at 70 °C for 2 min.
• High yield: Perform two elution steps with the volume indicated in the individual
protocol. About 90–100 % of bound nucleic acids can be eluted.
* Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor, Cold Spring,
New York 1982.

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• High concentration: Perform one elution step with 60 % of the volume
indicated in the individual protocol. Concentration of DNA will be higher than
with standard elution (approx. 130 %). Maximal yield of bound nucleic acids is
about 80 %.
• High yield and high concentration: Apply half of the volume of elution buffer
as indicated in the individual protocol, incubate for 3 min and centrifuge. Apply
a second aliquot of elution buffer, incubate, and centrifuge again. Thus, about
85–100 % of bound nucleic acids are eluted with the standard elution volume
at a high concentration.
Elution Buffer AE (5 mM Tris/HCl, pH 8.5) can be replaced by TE buffer or water as well.
However, we recommend using a weakly buffered, slightly alkaline buffer containing no
EDTA, especially if the plasmid DNA is intended for sequencing reactions. If water is
used, the pH should be checked and adjusted to pH 8.0–8.5 since deionized water
usually exhibits a pH below 7. Furthermore absorption of CO2leads to a decrease in
pH of unbuffered solutions.

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3 Storage conditions and preparation of working
solutions
Attention: Buffer A3 contains guanidine hydrochloride! Wear gloves and goggles!
CAUTION: Buffer A3 contains guanidine hydrochloride which can form highly reactive
compounds when combined with bleach (sodium hypochlorite). DO NOT add bleach or
acidic solutions directly to the sample-preparation waste.
• All kit components can be stored at room temperature (18–25 °C) and are
stable for at least one year.
• Always keep buffer bottles tightly closed, especially if buffers are preheated
during the preparation.
• Storage of Buffer A2 below 20 °C may cause precipitation of SDS. If salt
precipitate is observed, incubate buffer at 30–40 °C for several minutes and
mix well until all precipitate is redissolved completely. Cool down to room
temperature before use.
Before starting any NucleoSpin®Plasmid EasyPure protocol prepare the following:
• Add Liquid RNase A to Buffer A1 and mix thoroughly. Indicate date of RNase A
addition.StoreBufferA1containingRNaseAat4 °C.Thesolutionwillbestable
at this temperature for at least six months.
• Add the indicated volume of 96–100 % ethanol to Buffer AQ (Concentrate).
NucleoSpin®Plasmid EasyPure
10 preps 50 preps 250 preps
REF 740727.10 740727.50 740727.250
Wash Buffer AQ
(Concentrate)
6 mL
Add 24 mL ethanol
6 mL
Add 24 mL ethanol
30 mL
Add 120 mL ethanol