ISOLATE II

Plasmid Mini Kit

Product Manual
2 Product Manual www.bioline.com/isolate
 Plasmid Mini Kit

ISOLATE II Plasmid Mini Kit

ISOLATE II Plasmid Mini Kit

1 Kit contents 04
2 Description 04
3 Storage 04
4 Safety information 05
5 Product specifications 05
6 Equipment and reagents to be supplied by the user 07
7 Important notes 07
7.1 Growth of bacterial cultures 07
7.2 Buffer preparation and parameters 09
8 Protocol 09
8.1 Isolation of high‑copy plasmid DNA from E. coli 09
9 Alternative protocols 11
9.1 Isolation of low‑copy plasmid, P1 constructs or 11
cosmid DNA from E. coli
9.2 Isolation of plasmid DNA from Gram‑positive 12
bacteria
10 Troubleshooting guide 13
General Information
A Technical support 15
B Ordering information 15
C Associated products 15
D Product warranty and disclaimer 15

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 1. KIT CONTENTS

COMPONENT 10 Preps 50 Preps 250 Preps

ISOLATE II Plasmid Mini Spin Columns

10 50 250
(white)
Collection Tubes (2 mL) 10 50 250

Resuspension Buffer P1 5 mL 15 mL 75 mL

Lysis Buffer P2 5 mL 15 mL 100 mL

Neutralization Buffer P3 5 mL 20 mL 100 mL

Wash Buffer PW1 6 mL 30 mL 2 x 75 mL

Wash Buffer PW2† (concentrate) 6 mL 12 mL 2 x 25 mL

Elution Buffer P 13 mL 13 mL 60 mL

RNase A (lyophilized) 2.5 mg 6 mg 30 mg

Product Manual 1 1 1

Bench Protocol Sheet 1 1 1

†
Before use, add indicated volume of 96‑100% ethanol and mark wash buffer bottle label.

2. DESCRIPTION
The ISOLATE II Plasmid Mini Kit is a simple, reliable and fast method for isolation of
high‑quality plasmid DNA from E. coli host cells by SDS/alkaline lysis. The lysate is
subsequently neutralized and adjusted to high‑salt binding conditions in one step. After
lysate clearing, the sample is ready for purification on a silica membrane to which the plasmid
binds. Any contamination and impurities such as salts, metabolites and cellular components
are effectively removed by simple washing steps with two different buffers. High‑quality
purified plasmid is then eluted in an elution buffer.

Please read this manual carefully to familiarize yourself with the ISOLATE II Plasmid Mini
protocol before starting (also available on www.bioline.com). More experienced users can
refer to the bench‑top protocol for quick referencing during the procedure.

3. STORAGE
Store Resuspension Buffer P1 containing RNase A at 4°C (stable for at least 6 months). All
other kit components should be stored at room temperature (18–25°C) and are stable for up
to 1 year. Storage at lower temperatures may cause precipitation of salts. If a precipitate of
the SDS is observed in Lysis Buffer P2, incubate the bottle at 30–40°C for several minutes
and mix well. Always keep buffer bottles tightly closed, especially if buffers are pre‑heated
during the preparation.

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4. SAFETY INFORMATION
When working with chemicals, always wear a suitable lab coat, gloves and safety glasses.

Neutralization Buffer P3  and Wash Buffer PW1  contain guanidine hydrochloride. This
chemical is harmful when in skin contact, inhaled or ingested.

For detailed information, please consult the material data safety sheets (MSDSs) available on
our website at www.bioline.com.

5. PRODUCT SPECIFICATIONS
The ISOLATE II Plasmid Mini Kit is specially designed for the small‑scale rapid and efficient
isolation of extremely pure plasmid DNA. The Plasmid Mini Columns offers very high DNA
binding capacity of up to 60  µg, provided there is thorough washing, which is strongly
recommended for host strains with high levels of endonucleases like HB101 or JM110.

The ISOLATE II Plasmid Mini Kit allows purification of low‑copy plasmids from larger culture
volumes, purification of plasmids from Gram‑positive bacteria and clean‑up of plasmids from
reaction mixtures. The purified plasmid DNA is suitable for applications such as fluorescent
DNA sequencing, PCR and enzymatic manipulation.

ISOLATE II PLASMID MINI COLUMN SPECIFICATIONS

Max. binding capacity 60 μg plasmid DNA

1‑5 mL high copy
Culture volume
5‑10 mL low copy
<25 µg (1‑5 mL culture)
Typical yield
<40 µg (5‑10 mL culture)
Elution volume 50 μL

Max. plasmid size <15 kb

Hands on preparation time 25 min /18 preps

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 Plasmid DNA Isolation

Bacterial cell culture Wash silica membrane

1‑5 mL high copy 1st wash 500 µL Wash Buffer PW1 (optional)
plasmid DNA* 2nd wash 600 µL Wash Buffer PW2

11,000 x g, 30s
1st and 2nd
11,000 x g, 1 min
Cell lysis
Add 250 µL Resuspension Dry silica membrane
Buffer P1 11,000 x g, 2 min
Vortex

Add 250 µL Lysis Buffer P2

Invert 6‑8 times, incubate Elute DNA
at RT for 5 mins Add 50 µL Elution Buffer P
Incubate RT, 1 min
Add 300 µL Neutralization
Buffer P3
Invert 6‑8 times
11,000 x g, 1 min
Clear lysate
11,000 x g, 5‑10 min

Isolated Plasmid DNA

Bind DNA
Load supernatant

11,000 x g, 1 min

* For low copy plasmids, use 5‑10 mL bacterial culture and double volume of buffers P1,
P2 and P3 for lysis.

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6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER

When working with chemicals, always wear a suitable lab coat, protective goggles and
disposable gloves.

• 96‑100% ethanol†
• Microcentrifuge tubes (1.5 mL)
• Sterile DNase‑free tips
• Pipettes
• Microcentrifuge (capable of 11,000 x g)
• Vortex mixer
• Thermal heating block

†
Molecular biology grade ethanol is recommended. Do not use denatured alcohol which contains
unwanted additives such as methanol and acetone.

7. IMPORTANT NOTES
7.1 GROWTH OF BACTERIAL CULTURES
Plasmids are generally prepared from bacterial cultures grown in the presence of a selective
agent such as an antibiotic (Table 1). The yield and quality of plasmid DNA may depend on
factors such as plasmid copy number, host strain, inoculation, antibiotic and type of culture
medium.

TABLE 1: ANTIBIOTICS

Stock solution Working

Antibiotic* Storage
(concentration) concentration
Ampicillin 100 mg/mL in water ‑20°C 50‑200 µg/ mL

Carbenicillin 100 mg/mL in ethanol ‑20°C 20‑200 µg/ mL

Chloramphenicol 50 mg/mL in ethanol ‑20°C 25‑170 µg/ mL

Kanamycin 10 mg/mL in water ‑20°C 10‑50 µg/ mL

Neomycin 50 mg/mL in water ‑20°C 50 µg/ mL

Tetracycline 12.5 mg/mL in ethanol ‑20°C 12.5‑50 µg/ mL

* See associated products

Plasmids vary widely in their copy number per cell (Table 2), depending on their origin of
replication (e.g. ColE1, pMB1 or pSC101) which determines whether they are under relaxed or
stringent control. Also, depending on the size of the plasmid and its associated insert, overall
yield can be affected.

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 TABLE 2: ORIGINS OF REPLICATION AND COPY NUMBERS

Plasmid type* Origin of replication Copy number Classification

pTZ pMB1 >1000 High copy

pUC pMB1 500–700 High copy

pBluescript ColE1 300–500 High copy

pGEM® pMB1 300–400 High copy

pBR322 and
pMB1 15–20 Low copy
derivatives
pACYC and derivatives p15A 10–12 Low copy

pSC101 and derivatives pSC101 ~5 Very low copy

* See associated products

For cultivation of bacterial cells harbouring standard high‑copy plasmids, we recommend

Lysogeny Broth (LB) medium. Bacterial cultures for plasmid preparation should always be
grown from a single colony picked from a freshly streaked selective plate. The cell culture
should be incubated at 37°C with constant shaking (200‑250 rpm) overnight (12‑16 h), to give
an OD of 3‑6. Alternatively, rich media like 2 x YT (Yeast/Tryptone) or TB (Terrific Broth) can
be used. In this case bacteria grow faster, reach the stationary phase much sooner (≤12 h)
with a higher cell mass. However, this does not necessarily yield more plasmid DNA.

Growth for more than 16  hours (12  hours for rich media) is not recommended since cells
begin to lyse and plasmid yields may be reduced. This can also lead to contamination with
chromosomal DNA. To find the optimal culture conditions, the culture medium and incubation
times should be optimized for each host strain / plasmid construct combination individually.
As a general guide we recommend using 5 mL of a well grown culture (for more accurate
guide see Table 3).

TABLE 3: RECOMMENDED CULTURE VOLUMES ACCORDING TO THE OPTICAL DENSITY

OD600 1 2 3 4 5 6

Culture volume 15 mL 8 mL 5 mL 4 mL 3 mL 2 mL

Note: If excess culture volume is used, alkaline lysis will be inefficient, the membrane will be overloaded and
performance will decrease. If more than the recommended amount of cells shall be processed, refer to the support
protocol for low‑copy plasmid purification (section 9.1).

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7.2 BUFFER PREPARATION AND PARAMETERS

Preparing Resuspension Buffer P1
Add 1 mL of Resuspension Buffer P1 to the RNase A vial and vortex. Transfer all of the resulting
solution into the Resuspension Buffer P1  bottle and mix thoroughly. Store Resuspension
Buffer P1 containing RNase A at 4°C.
Note: The solution will be stable at this temperature for at least six months.

Preparing Wash Buffer PW2

Add 96–100% ethanol to Wash Buffer PW2 Concentrate: 8 mL for the 10 prep kit, 24 mL x
2 for the 50 prep kit and 80 mL x 2 for the 250 prep kit.

Alternative Elution Parameters

It is possible to modify the standard elution protocol to improve yield and concentration.
Use Elution Buffer P preheated to 70°C for one of the following procedures:
• High yield, especially for larger constructs: Heat Elution Buffer P to 70°C, add
50‑100 µL to the column and incubate at 70°C for 2 min.
• High yield: Two elution steps with 50 μL Elution Buffer P (to increase yield to 90–100%).
• High concentration: One elution step with 30  μL Elution Buffer P (to increase
concentration by about 130%). Maximal yield 80%.
• High yield and high concentration: Two elution steps. Add 25  μL Elution Buffer P,
incubate for 3 min and centrifuge, repeat with a second 25 μL Elution Buffer P. Yield
85–100% at a high concentration.

8. PROTOCOL
8.1 ISOLATION OF HIGH‑COPY PLASMID DNA FROM E. COLI
Before you start:
• Make sure Wash Buffer PW2 and Resuspension Buffer P1 are prepared (see section
7.2).

1 Harvest bacterial cells

Use 1‑5 mL of a saturated E. coli LB culture (see section 7.1), pellet cells for 30s at
11,000 x g. Discard the supernatant and remove as much liquid as possible.
Note: For isolation of low‑copy plasmids refer to section 9.1.

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 2 Lyse cells
• Add 250 μL Resuspension Buffer P1 and resuspend the cell pellet completely
by vortexing or pipetting up and down, making sure no cell clumps remain.
• Add 250 µL Lysis Buffer P2. Mix gently by inverting the tube 6‑8 times.
Note: Do not vortex to avoid shearing of genomic DNA.
Incubate at room temperature for up to 5 min or until lysate appears clear.
• Add 300  µL Neutralization Buffer P3. Mix thoroughly by inverting the tube
6‑8 times.
Note: Do not vortex to avoid shearing of genomic DNA.
3 Clarification of lysate
Centrifuge for 5 min at 11,000 x g at room temperature.
Repeat this step if supernatant is not clear.
4 Bind DNA
For each preparation, take one ISOLATE II Plasmid Mini Spin Column, placed in a
Collection Tube and decant or pipette a maximum of 750 µL of the clarified sample
supernatant onto the column. Centrifuge for 1  min at 11,000  x  g and discard
flow‑through.
Repeat with any remaining clarified sample supernatant.
5 Wash silica membrane
If plasmid DNA is prepared from host strains containing high levels of nucleases (e.g.
HB101 or strains of the JM series), we strongly recommend performing an additional
wash step at this point with Wash Buffer PW1.
(Optional) Add 500 µL Wash Buffer PW1 preheated to 50°C and centrifuge for 1 min
at 11,000 x g before proceeding.
Note: Additional washing with Wash Buffer PW1  will also increase the read length of DNA
sequencing reactions and improve the performance of critical enzymatic reactions.
Add 600 μL Wash Buffer PW2 (supplemented with ethanol) and centrifuge 1 min at
11,000 x g. Discard flow‑through and reuse Collection Tube.
6 Dry silica membrane
Centrifuge 2  min at 11,000  x  g, to remove residual ethanol. Place the ISOLATE II
Plasmid Mini Spin Column in a 1.5 mL microcentrifuge tube (not supplied).
7 Elute DNA
Add 50  μL Elution Buffer P directly onto the silica membrane. Incubate at room
temperature for 1 min. Centrifuge 1 min at 11,000 x g.
Note: For alternative elution procedures see section 7.2.

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9. ALTERNATIVE PROTOCOLS
9.1 ISOLATION OF LOW‑COPY PLASMID, P1 CONSTRUCTS OR COSMID DNA FROM
E. COLI
Processing of larger culture volumes requires increased lysis buffer volumes. The buffer
volumes provided with the kit are calculated for high‑copy plasmid purification only.

Before you start:

• Make sure Wash Buffer PW2 and Resuspension Buffer P1 are prepared (see section
7.2).

1 Harvest bacterial cells

Use 5‑10 mL of a saturated E. coli LB culture, pellet cells for 30s at 11,000 x g. Discard
the supernatant and remove as much liquid as possible.
2 Lyse cells
• Add 500 μL Resuspension Buffer P1 and resuspend the cell pellet completely
by vortexing or pipetting up and down, making sure no cell clumps remain.
• Add 500 µL Lysis Buffer P2. Mix gently by inverting the tube 6‑8 times.
Note: Do not vortex to avoid shearing of genomic DNA.
Incubate at room temperature for up to 5 min or until lysate appears clear.
• Add 600  µL Neutralization Buffer P3. Mix thoroughly by inverting the tube
6‑8 times.
Note: Do not vortex to avoid shearing of genomic DNA.
3 Clarification of lysate
Centrifuge for 10 min at 11,000 x g at room temperature.
4 Bind DNA
For each preparation, place one ISOLATE II Plasmid Mini Spin Column, in a Collection
Tube and decant or pipette 750  µL of the clarified sample supernatant onto the
column. Ensure all lysate is loaded.
Centrifuge for 1 min at 11,000 x g and discard flow‑through.
Repeat with any remaining clarified sample supernatant.
5 Wash silica membrane
(Optional) Add 500 µL Wash Buffer PW1 preheated to 50°C and centrifuge for 1 min
at 11,000 x g before proceeding.
Note: Additional washing with Wash Buffer PW1  will also increase the reading length of DNA
sequencing reactions and improve the performance of critical enzymatic reactions.
Add 600 μL Wash Buffer PW2 (supplemented with ethanol) and centrifuge 1 min at
11,000 x g. Discard flow‑through and reuse Collection Tube.

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 6 Dry silica membrane
Centrifuge 2  min at 11,000  x  g to remove residual ethanol. Place the ISOLATE II
Plasmid Mini Spin Column in a 1.5 mL microcentrifuge tube (not supplied).
7 Elute DNA
Add 50 μL Buffer P preheated to 70°C directly onto the silica membrane. Incubate for
2 min at 70°C. Centrifuge 1 min at 11,000 x g.
Note: For alternative elution procedures see section 7.2.

9.2 ISOLATION OF PLASMID DNA FROM GRAM‑POSITIVE BACTERIA

For plasmid purification from bacteria such as Bacillus or Staphylococcus, it is necessary
to start the lysis procedure with an enzymatic treatment (e.g. lysozyme, lysostaphin,
mutanolysin) to break up the peptidoglycan layers. For some Gram‑positive bacteria such
as Bifidobacteria or Corynebacteria, a preincubation with lysozyme might be insufficient and
mechanical cell disruption methods have to be used.

Before you start:

• Make sure Buffer PW2 and Resuspension Buffer P1 are prepared (see section 7.2).

1 Harvest bacterial cells

Use 1‑5 mL of a saturated E. coli LB culture, pellet cells for 30s at 11,000 x g. Discard
the supernatant and remove as much liquid as possible.
2 Lyse cells
• Add 250  μL Resuspension Buffer P1  containing 10  mg/mL lysozyme (not
supplied) and resuspend the cell pellet completely by vortexing or pipetting
up and down, making sure no cell clumps remain.
Incubate at 37°C for 10‑30 min.
• Add 250 µL Lysis Buffer P2. Mix gently by inverting the tube 6‑8 times.
Note: Do not vortex to avoid shearing genomic DNA.
Incubate at room temperature for up to 5 min or until lysate appears clear.
• Add 300  µL Neutralization Buffer P3. Mix thoroughly by inverting the tube
6‑8 times.
Note: Do not vortex to avoid shearing genomic DNA.
Proceed with step 3 of the standard protocol (see section 8.1).

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10. TROUBLESHOOTING GUIDE

INCOMPLETE LYSIS OF BACTERIAL CELLS

POSSIBLE CAUSE RECOMMENDED SOLUTION

Cell pellet not properly It is essential that the cell pellet is completely resuspended prior
resuspended to lysis. No cell clumps should be visible before addition of Lysis
Buffer P2.
Too many bacterial cells used We recommend LB as optimal growth medium. If very rich media
like TB (Terrific Broth) is used, the cell density of the cultures may
become too high and culture time should be reduced.
SDS in Lysis Buffer Sodium dodecyl sulfate (SDS) in Lysis Buffer P2 may precipitate
P2 precipitated if stored at temperatures below 20°C, redissolve by incubating
the bottle at 30‑40°C for several minutes and mixing well.

POOR PLASMID YIELD

POSSIBLE CAUSE RECOMMENDED SOLUTION

Incomplete lysis of bacterial cells Cell pellet not properly resuspended, too many bacterial cells
used or SDS in Lysis Buffer P2 precipitated, see above.
Suboptimal precipitation of SDS Precipitation of SDS and cell debris will be slightly more effective
and cell debris when centrifuging at 4°C rather than room temperature.
No or insufficient amounts of Add appropriate amounts of freshly prepared stock solutions to
antibiotic used during cultivation all media; both solid and liquid.
Bacterial culture too old Do not incubate cultures for >16 h at 37°C when using LB and
<12 h when using very rich media like TB (Terrific Broth).
Suboptimal elution conditions If not using Elution Buffer P, make sure elution buffer used is
slightly alkaline (pH 8.5). If nuclease‑free water is used, check
the pH of the water.
No high copy‑number plasmid If using low copy‑number plasmids, the culture volumes should
was used be increased to at least 5 mL.

NO PLASMID YIELD

POSSIBLE CAUSE RECOMMENDED SOLUTION

Reagents not applied properly Add indicated volume of 96‑100% ethanol to Wash Buffer
PW2 Concentrate and mix thoroughly (see section 7.2).
Inappropriate storage of plasmid Store plasmid DNA dissolved in water at <‑18°C or at 4°C when
DNA dissolved in Elution Buffer P or TE buffer.
Nuclease‑rich host strains used If using nuclease‑rich strains like E. coli HB101 or strains of the
JM series, keep plasmid preparations on ice or frozen in order
to avoid DNA degradation and perform the optional PW1 wash
step (step 5; section 8.1). Optimal endonuclease removal can
be achieved by incubating the membrane with preheated Wash
Buffer PW1 (50°C) for 2 min before centrifugation.

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 POOR PLASMID QUALITY

POSSIBLE CAUSE RECOMMENDED SOLUTION

Nicked plasmid DNA due to Cell suspension was incubated with alkaline Lysis Buffer P2 for
excessive incubation with Lysis too long (more than 5 min), incubate for a shorter time.
Buffer P2
Genomic DNA contamination Cell lysate was vortexed or mixed too vigorously after addition of
Lysis Buffer P2, shearing the genomic DNA.
Smeared plasmid bands on If using nuclease‑rich strains like E. coli HB101 or strains of the
agarose gel JM series, keep plasmid preparations on ice or frozen in order to
avoid DNA degradation and perform the optional PW1 washing
step (step 5; section 8.1). Optimal endonuclease removal can
be achieved by incubating the membrane with preheated Wash
Buffer PW1 (50°C) for 2 min before centrifugation.

SUBOPTIMAL PERFORMANCE OF PLASMID DNA IN ENZYMATIC REACTIONS

POSSIBLE CAUSE RECOMMENDED SOLUTION

Carry‑over of ethanol Make sure to centrifuge ≥1 min at 11,000 x g in step 6 to achieve
total removal of Wash Buffer PW2.
Elution of plasmid DNA with TE EDTA may inhibit sequencing reactions. Repurify plasmid DNA
buffer and elute with Elution Buffer P or water. Alternatively precipitate
with ethanol and redissolve in Elution Buffer P or water.
No additional washing with Wash Additional washing with 500 µL Wash Buffer PW1 (step 5; section
Buffer PW1 performed 8.1) will increase the reading length of sequencing reactions and
improve the performance of critical enzymatic reactions.
Not enough DNA used for Quantitate DNA on an agarose gel before setting up sequencing
sequencing reaction reactions.
Plasmid DNA prepared from too Do not use more than 3 mL of a saturated E. coli culture if
much bacterial cell material preparing plasmid DNA for DNA sequencing.

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A. TECHNICAL SUPPORT
For technical assistance or more information on these products, please email us at
[email protected]

B. ORDERING INFORMATION

PRODUCT PACK SIZE CAT NO.

ISOLATE II Plasmid Mini Kit 10 Preps BIO‑52055

ISOLATE II Plasmid Mini Kit 50 Preps BIO‑52056

ISOLATE II Plasmid Mini Kit 250 Preps BIO‑52057

C. ASSOCIATED PRODUCTS

PRODUCT PACK SIZE CAT NO.

a‑Select Gold Efficiency 1 mL (20 x 50 μL) BIO‑85027

Agarose 100 g BIO‑41026

IPTG 5g BIO‑37036

X‑Gal 1g BIO‑37035
100 Reactions (10
Quick‑Stick Ligase BIO‑27028
u/μL)

D. PRODUCT WARRANTY AND DISCLAIMER

Bioline warrants that its products will conform to the standards stated in its product
specification sheets in effect at the time of shipment. Bioline will replace free of charge any
product that does not conform to the specifications. This warranty limits Bioline’s liability
only to the replacement of the product.

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