QMF27.0085.D6943 v5.3

E.Z.N.A.
® Plasmid DNA Mini Kit I

D6942-00 5 preps Q - Spin
D6943-00 5 preps V - Spin

E.Z.N.A.® Plasmid DNA Mini Kit II

Manual Date: February 2022

Revision Number: v5.3
E.Z.N.A.® Plasmid DNA Mini Kit I
E.Z.N.A.® Plasmid DNA Mini Kit II
Table of Contents
Introduction......................................................................................................2
Yield and Quality of DNA............................................................................3
Illustrated Protocol.........................................................................................4
Kit Contents.......................................................................................................5
Preparing Reagents........................................................................................6
Storage and Stability......................................................................................7
Guidelines for Vacuum Manifold...............................................................8
Recommended Settings.........................................................................9-10
Plasmid DNA Mini Kit I Spin Protocol...............................................11
Plasmid DNA Mini Kit I Vacuum Protocol.......................................14
Plasmid DNA Mini Kit II Spin Protocol..............................................18
Low Copy Number Plasmid Protocol....................................................22
Troubleshooting Guide...............................................................................23
Manual Date: Feburary 2022

Revision Number: v5.3
1
Introduction
The E.Z.N.A.® family of products is an innovative system that radically simplifies the
extraction and purification of nucleic acids from a variety of sources. The key to this
system is the HiBind® matrix that specifically, but reversibly, binds DNA or RNA under
optimized conditions allowing proteins and other contaminants to be removed. Nucleic
acids are easily eluted with deionized water or low salt buffer.
The E.Z.N.A.® Plasmid DNA Mini Kits combine the power of HiBind® technology with the
time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high-quality DNA
in less than 30 minutes. HiBind® DNA Mini Columns facilitate the binding, washing, and
elution steps thus enabling multiple samples to be processed simultaneously.
Typically, a 1.5 mL overnight culture in LB medium produces 3-12 μg plasmid DNA;

although yields may vary according to plasmid copy number, E. coli strain, and growth
conditions. The E.Z.N.A.® Plasmid DNA Mini Kit I is used to isolate plasmid DNA from
1-5 mL cultures. The E.Z.N.A.® Plasmid DNA Mini Kit II can isolate 40-75 μg plasmid DNA
from 10-15 mL cultures when using high copy plasmids. Purified plasmid DNA can be
directly used for most downstream applications including automated fluorescent DNA
sequencing and restriction enzyme digestion.
Q-spin column vs. V-spin column
The E.Z.N.A.® Plasmid DNA Mini Kit is available with two different types of columns.
V-spin columns have an attached cap, while Q-spin columns are capless. The columns are
otherwise identical in use and application. Both columns can be used with the vacuum or
centrifugation protocol.
Protocols
The E.Z.N.A.® Plasmid DNA Mini Kits are designed for fast and efficient processing.
Depending on the protocol, the E.Z.N.A.® Plasmid Mini Kits can be used with any
microcentrifuge or vacuum manifold with standard luer connectors.
New In this Edition
Febuary 2022
• A new 600 prep kit has been added and is now available for purchase.
2
Yield and Quality of DNA
Determine the absorbance of an appropriate dilution (20- to 50-fold) of the sample at

260 nm and then at 280 nm. The DNA concentration is calculated as follows:
DNA concentration = Absorbance 260 × 50 × (Dilution Factor) µg/mL
A ratio greater than 1.8 indicates greater than 90% nucleic acid. Alternatively, quantity
(as well as quality) sometimes can be determined best by agarose gel/ethidium bromide
electrophoresis by comparison to DNA samples of known concentrations. Typically, the
majority of the DNA eluted is in monomeric supercoil form, though concatemers may also
be present.
Plasmid Copy Number and Expected Yield
Yield and quality of the plasmid DNA obtained depends on a number of factors including
plasmid copy number, size of insert, host strain, culture volume, culture medium, and
binding capacity of the kit. Of these factors, the vector copy number, culture volume,
and kit binding capacity are most important. Plasmid copy number ranges from one
copy to several hundred copies per cell as dictated by their origin of replication. Very
large plasmids often display a very low copy number per cell. The expected yield of 5 mL
overnight cultures (LB medium) with the E.Z.N.A.® Plasmid Mini Kit are indicated in the
following table.
Sample yields from a 5 mL starting culture.

Plasmid Replicon Copy Number Expected Yield
pUC vectors pMBI 500-700 15-25 µg
pBluescript® vectors ColE14 300-500 10-18 µg
pGEM® vectors pMB1 300-400 10-20 µg
pBR322 and its derivatives pMB1 15-20 1-2 µg
ColE14 ColE14 15-20 1-2 µg
PACYC and its derivatives p15A 37540 0.5-1 µg
pSC101 and its derivatives pSC101 ~5 0.5 µg
pGEM pMB1 300-700 10-20 µg
3
Spin Protocol Vacuum/Spin Protocol
Pellet by Pellet by
Centrifugation Centrifugation
Resuspend Resuspend
and Lyse and Lyse
Neutralize Neutralize
Clear Lysate Clear Lysate
Transfer Lysate to Transfer Lysate to

HiBind® DNA Mini Column HiBind® DNA Mini Column
Bind Bind
Wash 3X Wash 3X
Dry Dry
Elute Elute
4
Kit Contents
D6942-00 D6942-01 D6942-02 D6942-03

Plasmid Mini Kit I
D6943-00 D6943-01 D6943-02 D6943-03
Preps 5 50 200 600
HiBind® DNA Mini Columns 5 50 200 600
2 mL Collection Tubes 5 50 200 600
Solution I 3 mL 20 mL 60 mL 200 mL
Solution II 3 mL 20 mL 60 mL 200 mL
Solution III 3 mL 20 mL 80 mL 250 mL
HBC Buffer 5 mL 25 mL 80 mL 250 mL
DNA Wash Buffer 2.5 mL 25 mL 3 x 25 mL 100 mL
RNase A Pre-Added 100 µL 400 µL 1.5 mL
Elution Buffer 2 mL 15 mL 30 mL 100 mL
User Manual    
Plasmid Mini Kit II D6945-00 D6945-01 D6945-02
Preps 5 50 200
HiBind® DNA Mini Columns II 5 50 200
2 mL Collection Tubes 5 50 200
Solution I 5 mL 30 mL 120 mL
Solution II 5 mL 30 mL 120 mL
Solution III 5 mL 40 mL 2 x 80 mL
HBC Buffer 5 mL 25 mL 80 mL
DNA Wash Buffer 2.5 mL 25 mL 3 x 25 mL
RNase A Pre-Added 100 µL 400 µL
Elution Buffer 2 mL 30 mL 30 mL
User Manual   
5
Preparing Reagents
1. Add the vial of RNase A to the bottle of Solution I and store at 2-8˚C. (50, 200 and 600
prep size only).
2. Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.
Kit 100% Ethanol to be Added

D6942-00
D6943-00 10 mL
D6945-00
D6942-01
D6943-01 100 mL
D6945-01
D6942-02
D6943-02 100 mL
D6945-02
D6942-03
400 mL
D6943-03
3. Dilute HBC Buffer with 100% isopropanol as follows and store at room temperature.
Kit 100% Isopropanol to be Added

D6942-00
D6943-00 2 mL
D6945-00
D6942-01
D6943-01 10 mL
D6945-01
D6942-02
D6943-02 32 mL
D6945-02
D6942-03
100 mL
D6943-03
6
Storage and Stability
All of the E.Z.N.A.® Plasmid DNA Mini Kit I and Plasmid DNA Mini Kit II components are
guaranteed for at least 12 months from the date of purchase when stored as follows.
Solution I (once RNase A is added) should be stored at 2-8˚C. All other materials should be
stored at room temperature. Solution II must be tightly capped when not in use. During
shipment or storage in cool ambient conditions, precipitates may form in some buffers.
Dissolve such deposits by warming the solution at 37˚C and gently shaking.
7
Guidelines for Vacuum Manifold
The following is required for use with the Vacuum/Spin Protocol:
A) Vacuum Manifold
Compatible Vacuum Manifolds: Qiagen QIAvac24, Sigma Aldrich VM20,
Promega Vacman®, or manifold with standard Luer connector
B) Vacuum Flask
C) Vacuum Tubing
D) Vacuum Source (review tables below for pressure settings)
Conversion from millibars: Multiply by:

Millimeters of mercury (mmHg) 0.75
Kilopascals (kPa) 0.1
Inches of mercury (inch Hg) 0.0295
Torrs (Torr) 0.75
Atmospheres (atmos) 0.000987
Pounds per Square Inch (psi) 0.0145
Vacuum Setup:
Vacuum Manifold
C) Vacuum Tubing
D) Vacuum Source
A) Vacuum Manifold
B) Vacuum Flask
8
Recommended Settings
Growth and Culture of Bacteria
Bacterial Strain Selection
It is strongly recommended that an end A negative strain of E. coli be used for routine
plasmid isolation. Examples of such strains include DH5α™ , DH1, and C600. These host
strains yield high-quality DNA with E.Z.N.A.® Plasmid DNA Mini Kit Protocols. XL1-Blue,
although a slower growing strain is also recommended due to its yield of high-quality
DNA.
Host strains derivatives from HB101 such as TG1 and the JM100 series release large
amounts of carbohydrates during lysis, which may inhibit enzyme activity when not
completely removed. Some strains may also lower DNA quality due to having high levels
of endonuclease activity, and therefore are not recommended (i.e. JM101, JM110, HB101).
One may reduce the amount of culture volume or double the volumes of Solution I,
Solution II, and Solution III, if problems are encountered with strains such as TG1 and
Top10F.
Inoculation
Bacterial cultures for plasmid preparations should always be grown from a single colony
picked from a freshly streaked plate. Subculturing directly from glycerol stock or liquid
cultures may lead to uneven yields or plasmid loss. Optimal results are obtained by using
one single isolated colony from a freshly transformed or freshly streaked plate to inoculate
an appropriate volume of starter culture containing the appropriate antibiotic, and then
incubated for 12-16 hours at 37°C with vigorous shaking (~300 rpm; shaking incubator).
Note: Aeration is very important. The culture volume should not exceed 1/4 the volume of
the container.
Culture Media
The E.Z.N.A.® Plasmid DNA Mini Kits are specially designed for use with cultures grown
in Luria Bertani (LB) medium. Richer broths such as TB (Terrific Broth) or 2xYT lead to
high cell densities that can overload the purification system, and therefore are not
recommended. If rich media has to be used, growth times have to be optimized, and the
recommended culture volumes must be reduced to match the capacity of the HiBind®
DNA Mini Column.
Note: As culture ages, DNA yield may begin to decrease due to cell death and lysis within
the culture.
9
Recommended Settings
Culture Volume and Cell Density

Do Not Exceed Maximum Recommended Culture Volumes
For optimal plasmid yields, the starting culture volume should be based on culture cell
density. A bacterial density between 2.0 and 3.0 at OD600 is recommended. When using
nutrient-rich media, care should be taken to ensure that the cell density does not exceed
an OD600 of 3.0. Using a high-density culture outside of the recommended OD range may
overload the purification system.
10
E.Z.N.A.® Plasmid DNA Mini Kit I Spin Protocol
E.Z.N.A.® Plasmid DNA Mini Kit I Protocol - Spin Protocol

All centrifugation should be performed at room temperature unless otherwise noted. For
low copy number plasmids refer to Page 22. This protocol is designed to isolate plasmid
DNA from E. coli grown in an overnight 1-5 mL LB culture.
Materials and Equipment to be Supplied by User:
• 100% ethanol
• 100% isopropanol
• Microcentrifuge capable of at least 13,000g
• Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes
• Culture tubes
• Optional: sterile deionized water
• Optional: water bath or incubator capable of 70°C
• Optional: 3M NaOH solution
Before Starting:
• Heat Elution Buffer to 70°C if plasmid DNA is >10 kb

• Prepare DNA Wash Buffer, HBC Buffer, and Solution I according to the instructions in
the “Preparing Reagents” section on Page 6
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture
of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for
~12-16 hours at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube
or a flask with a volume of at least 4 times the volume of the culture. It is strongly
recommended that an endA negative strain of E. coli be used for routine plasmid
isolation. Examples of such strains include DH5α® and JM109®.
2. Centrifuge at 10,000g for 1 minute at room temperature.
3. Decant or aspirate and discard the culture media.
4. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.
Complete resuspension of cell pellet is vital for obtaining good yields.
Note: RNase A must be added to Solution I before use. Please see the instructions in
the “Preparing Reagents” section on Page 6.
11
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
6. Add 250 µL Solution II. Invert and gently rotate the tube several times to obtain a
clear lysate. A 2-3 minute incubation may be necessary.
Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid
purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution
II tightly capped when not in use to avoid acidification from CO2 in the air.
7. Add 350 µL Solution III. Immediately invert several times until a flocculent white
precipitate forms.
Note: It is vital that the solution is mixed thoroughly and immediately after the
addition of Solution III to avoid localized precipitation.
8. Centrifuge at maximum speed (≥13,000g) for 10 minutes. A compact white pellet will
form. Promptly proceed to the next step.
9. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
Optional Protocol for Column Equilibration:
1. Add 100 µL 3M NaOH to the HiBind® DNA Mini Column.

2. Centrifuge at maximum speed for 30-60 seconds.
3. Discard the filtrate and reuse the collection tube.
10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the
HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular
debris is transferred to the HiBind® DNA Mini Column.
11. Centrifuge at maximum speed for 1 minute.
13. Add 500 µL HBC Buffer.
Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page
6 for instructions.
12
15. Discard the filtrate and reuse collection tube.
16. Add 700 µL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 6 for instructions.
Optional: Repeat Steps 16-18 for a second DNA Wash Buffer wash step.
19. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to
dry the column matrix.
Note: It is important to dry the HiBind® DNA Mini Column matrix before elution.
Residual ethanol may interfere with downstream applications.
20. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
21. Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the
column membrane.
Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent
on pH. If using sterile deionized water, make sure that the pH is around 8.5.
22. Let sit at room temperature for 1 minute.
Note: This represents approximately 70% of bound DNA. An optional second elution
will yield any residual DNA, though at a lower concentration.
24. Store DNA at -20°C.

13
E.Z.N.A.® Plasmid DNA Mini Kit I Vacuum Protocol
E.Z.N.A.® Plasmid Mini Kit I Protocol - Vacuum Protocol

DNA from E. coli grown in an overnight 1-5 mL LB culture. See Page 8 for guidelines on
preparing the vacuum manifold used in this protocol.
• Vacuum manifold
• 100% ethanol
• Nuclease-free 1.5 mL or 2 mL microcentrifuge tubes
• Appropriate centrifuge and centrifuge tube for Step 1
Before Starting:
• Heat Elution Buffer to 70°C if plasmid DNA is >10 kb

of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for
~12-16 hr at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube
or a flask with a volume of at least 4 times the volume of the culture. It is strongly
recommended that an endA negative strain of E. coli be used for routine plasmid
isolation. Examples of such strains include DH5α® and JM109®.
2. Centrifuge at 10,000g for 1 minute at room temperature.
14
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
precipitate forms.
8. Centrifuge at maximum speed (≥13,000g) for 10 minutes. A compact white pellet will
form. Promptly proceed to the next step.
9. Prepare the vacuum manifold according to manufacturer’s instructions.
10. Connect the HiBind® DNA Mini Column to the vacuum manifold.

2. Turn on the vacuum source to draw the NaOH through the column.
3. Turn off the vacuum.
11. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the
HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular
debris is transferred to the HiBind® DNA Mini Column.
15
12. Turn on the vacuum source to draw the sample through the column.
6 for instructions.
15. Turn on the vacuum source to draw the buffer through the column.
18. Turn on the vacuum source to draw the buffer through the column.
20. Repeat Steps 17-19 for a second DNA Wash Buffer wash step.
21. Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube.
16
column membrane.
17
E.Z.N.A.® Plasmid DNA Mini Kit II Spin Protocol
E.Z.N.A.® Plasmid Mini Kit II Protocol - Spin Method

DNA from E. coli grown in 10-15 mL LB culture.
• 100% ethanol
• Nuclease-free 2 mL microcentrifuge tubes
• Centrifuge capable of at least 5,000g with swing buckets
• Appropriate centrifuge tube for Step 1
Before Starting:
• Heat Elution Buffer to 70°C if plasmid DNA is >10kb

of 10- 15 mL(50 µg/mL) LB medium containing the appropriate selective antibiotic.
Incubate for ~ 12-16 hours at 37°C with vigorous shaking (~ 300 rpm). Use culture
tube or a flask with a volume of at least 4 times the volume of the culture. It is
strongly recommended that an end A negative strain of E. coli be used for routine
plasmid isolation. Examples of such strains include DH5α® and JM109®.
2. Centrifugation at 5,000g for 10 minutes at room temperature.
3. Decant or aspirate the medium and discard.
18
5. Transfer suspension into a new 2 mL microcentrifuge tube.
precipitate forms.
8. Centrifuge at maximum speed (≥13,000g) for 10 minutes at room temperature. A

compact white pellet will form. Promptly proceed to the next step.
9. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.

2. Centrifuge at maximum speed for 30-60 seconds.
10. Transfer 700 µL cleared lysate from Step 8 by CAREFULLY aspirating it into the HiBind®
DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is
transferred to the HiBind® DNA Mini Column.
19
13. Repeat Steps 10-12 until all cleared lysate has been transferred to the HiBind® DNA
Mini Column.
6 for instructions.
16. Discard the filtrate and reuse collection tube.
Optional: Repeat Steps 17-19 for a second DNA Wash Buffer wash step.
20
column membrane.
21
Low Copy Number Plasmid and BAC DNA Protocol
E.Z.N.A.® Plasmid Mini Kit Protocol - Low Copy Number Plasmid

and BAC DNA Protocol
Low copy number plasmids generally give 0.1-1 µg DNA per mL overnight culture. For the
isolation of plasmid DNA from low copy number plasmids (0.1-1 µg/mL culture) or low
copy number plasmid (1-2 µg/mL culture) bacteria, use the following modified protocol.
Note: The E.Z.N.A.® Plasmid DNA Mini Kit l and the E.Z.N.A.® Plasmid DNA Mini Kit ll come
with enough Solution I, Solution II, and Solution III to perform the standard protocols.
Additional Solution I, Solution II, and Solution III are needed to perform the Low Copy
Number Plasmid and BAC DNA Protocol. These buffers can be purchased separately.
1. Increase the volume of starting culture from that of high copy number plasmids.
Use 5-10 mL bacterial culture for the E.Z.N.A.® Plasmid DNA Mini Kit l or 20-30 mL
bacterial culture for E.Z.N.A.® Plasmid DNA Mini Kit ll.
2. Pellet the bacterial cells by centrifugation.
4. Perform Steps 4-8 in the standard protocols with double the volumes of Solution I,
Solution II, and Solution III.
5. Continue with Step 9 of the standard protocols by following the wash, drying, and
elution steps. There is no need to increase the volumes of HBC Buffer, DNA Wash
Buffer, or Elution Buffer.
22
Troubleshooting Guide
Please use this guide to troubleshoot any problems that may arise. For further assistance,
please contact the technical support staff, toll free, at 800-832-8896.
Possible Problems and Suggestions
Low DNA yields
Only use LB or YT medium containing ampicillin. Do not use

more than 5 mL (high copy number plasmids) or 10 mL (low
copy number plasmids) culture with the basic protocols.
Cells may not have been dispersed adequately prior to the

Poor cell lysis
addition of Solution II. Vortex to completely resuspend the cells.
Increase Solution II incubation time to obtain a clear lysate.
Solution II, if not tightly closed, may need to be replaced.
Do not incubate cultures for more than 16 hours at 37ºC.

Culture is overgrown
Storage of cultures for extended periods prior to plasmid
or not fresh
isolation is detrimental.
Low elution efficiency The pH of Elution Buffer or water must be pH 8.0-9.0.
Such plasmids may yield as little as 0.1 μg DNA from a 1 mL

Low copy number
overnight culture. Double the culture volume and follow the
plasmid used
low copy number plasmid protocol on Page 22.
Follow the Optional Protocol for Column Equilibration prior
Column matrix lost to transferring the cleared lysate to the HiBind® DNA Mini
binding capacity Column. Add 100 µL 3M NaOH to the column prior to loading
during storage the sample. Centrifuge at 10,000g for 30 seconds. Discard the
filtrate.
No DNA eluted
DNA Wash Buffer not
Prepare DNA Wash Buffer according to instructions on Page 6.
diluted with ethanol
HBC Buffer not
diluted with Prepare HBC Buffer according to instructions on Page 6.
isopropanol
23
Troubleshooting Guide
High molecular weight DNA contamination of product

Over mixing of cell
lysate upon addition Do not vortex or mix aggressively after adding Solution II.
of Solution II
Overgrown cultures contain lysed cells and degraded DNA. Do

Culture overgrown
not grow cell longer than 16 hours.
Plasmid DNA floats out of well while loading agarose gel

Ethanol was not
completely removed Centrifuge column as instructed to dry the column before
from column elution.
following wash steps
Absorbance of purified DNA does not accurately reflect quantity of the

plasmid (A260/A280 ratio is high or low)
Check the absorbance of the ethanol between 250 nm and

DNA Wash Buffer is
300 nm. Do not use ethanol with high absorbance. Traces of
diluted with ethanol
impurities may remain on the binding column after washing
containing impurities
and contribute to the absorbance in the final product.
Plasmid DNA is
Confirm that the RNase A Solution was added to Solution I prior
contaminated
to first use. The RNase A solution may degrade due to high
with RNA; RNase
temperatures (>65 °C) or prolonged storage (> 6 months at
A treatment is
room temperature).
insufficient
Background reading
Spin the DNA sample at maximum speed for 1 minute; use the
is high due to silica
supernatant to repeat the absorbance readings.
fine particulates
Purification is
incomplete due to Reduce the initial volume of culture.
column overloading
24

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E.Z.N.A.

® Plasmid DNA Mini Kit I

D6943-00 5 preps V - Spin

E.Z.N.A.® Plasmid DNA Mini Kit II

Manual Date: February 2022

Manual Date: Feburary 2022

Typically, a 1.5 mL overnight culture in LB medium produces 3-12 μg plasmid DNA;

Q-spin column vs. V-spin column

New In this Edition

Determine the absorbance of an appropriate dilution (20- to 50-fold) of the sample at

DNA concentration = Absorbance 260 × 50 × (Dilution Factor) µg/mL

Plasmid Copy Number and Expected Yield

Sample yields from a 5 mL starting culture.

Clear Lysate Clear Lysate

Transfer Lysate to Transfer Lysate to

D6942-00 D6942-01 D6942-02 D6942-03

Plasmid Mini Kit II D6945-00 D6945-01 D6945-02

Kit 100% Ethanol to be Added

Kit 100% Isopropanol to be Added

The following is required for use with the Vacuum/Spin Protocol:

Conversion from millibars: Multiply by:

Growth and Culture of Bacteria

Bacterial Strain Selection

Culture Volume and Cell Density

E.Z.N.A.® Plasmid DNA Mini Kit I Protocol - Spin Protocol

Materials and Equipment to be Supplied by User:

• Heat Elution Buffer to 70°C if plasmid DNA is >10 kb

2. Centrifuge at 10,000g for 1 minute at room temperature.

3. Decant or aspirate and discard the culture media.

5. Transfer suspension into a new 1.5 mL microcentrifuge tube.

9. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.

Optional Protocol for Column Equilibration:

1. Add 100 µL 3M NaOH to the HiBind® DNA Mini Column.

11. Centrifuge at maximum speed for 1 minute.

12. Discard the filtrate and reuse the collection tube.

13. Add 500 µL HBC Buffer.

14. Centrifuge at maximum speed for 1 minute.

15. Discard the filtrate and reuse collection tube.

16. Add 700 µL DNA Wash Buffer.

17. Centrifuge at maximum speed for 1 minute.

18. Discard the filtrate and reuse the collection tube.

22. Let sit at room temperature for 1 minute.

23. Centrifuge at maximum speed for 1 minute.

24. Store DNA at -20°C.

E.Z.N.A.® Plasmid Mini Kit I Protocol - Vacuum Protocol

Materials and Equipment to be Supplied by User:

• Heat Elution Buffer to 70°C if plasmid DNA is >10 kb

2. Centrifuge at 10,000g for 1 minute at room temperature.

3. Decant or aspirate and discard the culture media.

5. Transfer suspension into a new 1.5 mL microcentrifuge tube.

9. Prepare the vacuum manifold according to manufacturer’s instructions.

Optional Protocol for Column Equilibration:

1. Add 100 µL 3M NaOH to the HiBind® DNA Mini Column.

13. Turn off the vacuum.

14. Add 500 µL HBC Buffer.

16. Turn off the vacuum.

17. Add 700 µL DNA Wash Buffer.

19. Turn off the vacuum.

21. Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube.

25. Let sit at room temperature for 1 minute.