Promega A1270 Wizard Plus SV Minipreps DNA Purification System Instruction Manual

Promega A1270 Wizard Plus SV Minipreps DNA Purification System

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Description

The Wizard® Plus SV Minipreps DNA Purification System provides a simple, reliable method for rapid isolation of plasmid DNA (Figure 1). The entire miniprep procedure can be completed in 30 minutes or less, depending on the number of samples processed. This system can be used to isolate any plasmid from E. coli hosts but works most efficiently when the plasmid is less than 20,000bp in size. Purified plasmids can be used without further manipulation for automated fluorescent DNA sequencing as well as for other standard molecular biology techniques. When used for in vitro transcription reactions, the isolated plasmid DNA should be supplemented with a ribonuclease inhibitor such as Recombinant RNasin® Ribonuclease Inhibitor (Cat.# N2511).
The protocol presented in this Technical Bulletin is for isolation of plasmid DNA from E. coli. Plasmid DNA can be purified from 1–10ml overnight cultures with the Wizard® Plus SV Minipreps DNA Purification System. Plasmid yield will vary depending on a number of factors, including culture volume, plasmid copy number, type of culture medium and the bacterial strain used.

Figure 1. Flow diagram of plasmid DNA isolation and purification using the Wizard® Plus SV Minipreps DNA Purification System.

Product Components and Storage Conditions

Storage Conditions: Store all Wizard® Plus SV Minipreps components at room temperature (15–30°C). Reagent expiration dates are listed on the product label.

Protocols

Do not exchange or replace components of the Wizard® Plus SV Minipreps DNA Purification System with components from any other Wizard® Plus System. Components from the Wizard® Plus and Wizard® Plus SV Systems are not interchangeable.

Materials to Be Supplied by the User
(Solution compositions are provided in Section 6.)

• LB agar plates containing antibiotic
• LB medium containing antibiotic
• ethanol (95%)
• microcentrifuge capable of 14,000 × g
• sterile 1.5ml microcentrifuge tubes
• centrifuge capable of 10,000 × g

Prior to using a new Wizard® Plus SV Minipreps DNA Purification System, dilute the provided Column Wash Solution (CWA) as follows:

• Add 7ml of 95% ethanol for a final volume of 11ml for the 10-prep system (Cat.# A1270).
• Add 35ml of 95% ethanol for a final volume of 55ml for the 50-prep system (Cat.# A1330 and A1340).
• Add 170ml of 95% ethanol for a final volume of 270ml for the 250-prep system (Cat.# A1460 and A1470) and 1,000-prep system (Cat.# A1465).

Preparation of E. coli

1. Use a single, well isolated colony from a fresh Luria-Bertani (LB) agar plate (containing antibiotic) to inoculate 1–10ml of LB medium (containing the same antibiotic). We recommend LB culture medium. Rich media, such as Terrific Broth, produce high cell densities that may overload the DNA purification system.
2. Incubate overnight (12–16 hours) at 37°C in a shaking incubator. Incubation time can be optimized to increase the plasmid DNA yield for a given culture volume. However, it has been observed that as a culture ages the DNA yield may begin to decrease due to cell death and lysis within the culture.

Note: An A600 reading of 2–4 ensures that cells have reached the proper growth density for harvesting and plasmid DNA isolation.

For high-copy-number plasmids , do not process more than 5ml of bacterial culture. If more than 5ml of culture is processed, the capacity of the Wizard® SV Minicolumn will be exceeded and no increase in plasmid yield will be obtained.

For low-copy-number plasmids, it may be necessary to process larger volumes of bacterial culture (up to 10ml) for recovery of sufficient DNA. Processing greater than 10ml of culture will lead to insufficient clearing of the bacterial lysate and thus increased contaminants in the plasmid DNA.

Production of a Cleared Lysate

Note: Throughout the remainder of this document, the supplied Cell Resuspension Solution (CRA), Cell Lysis Solution (CLA), Neutralization Solution (NSB) and Column Wash Solution (CWA) are referred to as Cell Resuspension Solution, Cell Lysis Solution, Neutralization Solution and Column Wash Solution, respectively.

1. Harvest 1–5ml (high-copy-number plasmid) or 10ml (low-copy-number plasmid) of bacterial culture by centrifuging for 5 minutes at 10,000 × g in a tabletop centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media.

2. Add 250μl of Cell Resuspension Solution and completely resuspend the cell pellet by vortexing or pipetting. It is essential to thoroughly resuspend the cells. If they are not already in a microcentrifuge tube, transfer the resuspended cells to a sterile 1.5ml microcentrifuge tube(s).
   Note: To prevent shearing of chromosomal DNA, do not vortex after Step 2. Mix only by inverting the tubes.

3. Add 250μl of Cell Lysis Solution and mix by inverting the tube 4 times (do not vortex). Incubate until the cell
   suspension clears (approximately 1–5 minutes).
   Note: It is important to observe partial clearing of the lysate before adding the Alkaline Protease Solution (Step 4);
   however, do not incubate for longer than 5 minutes.

4. Add 10μl of Alkaline Protease Solution and mix by inverting the tube 4 times. Incubate for 5 minutes at room temperature.
   Alkaline protease inactivates endonucleases and other proteins released during the lysis of the bacterial cells that can adversely affect the quality of the isolated DNA. Do not exceed 5 minutes of incubation with Alkaline Protease Solution at Step 4, as nicking of the plasmid DNA may occur.

5. Add 350µl of Neutralization Solution and immediately mix by inverting the tube 4 times (do not vortex).

6. Centrifuge the bacterial lysate at maximum speed (around 14,000 × g) in a microcentrifuge for 10 minutes at room temperature.

Plasmid DNA Isolation and Purification Protocols
The Wizard® Plus SV Minipreps DNA Purification System allows a choice of methods for purification of plasmid DNA when systems with Vacuum Adapters are purchased (Cat.# A1340, A1470). Plasmid DNA may be purified from the bacterial lysate using microcentrifugation to force the cleared lysate through the Wizard® SV Minicolumn and wash the plasmid DNA. Alternatively, a vacuum can be used to pull the lysate through the Spin Column and wash the plasmid DNA. Vacuum Adapters allow the use of a vacuum manifold (e.g., a Vac-Man® Laboratory Vacuum Manifold) and vacuum source for DNA purification.

Centrifugation Protocol
Prepare plasmid DNA purification units by inserting one Spin Column into one 2ml Collection Tube for each sample.

1. Transfer the cleared lysate (approximately 850µl, Section 3.B, Step 6) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant.
   Note: If the white precipitate is accidentally transferred to the Spin Column, pour the Spin Column contents back into a sterile 1.5ml microcentrifuge tube and centrifuge for another 5–10 minutes at maximum speed. Transfer the resulting supernatant into the same Spin Column that was used initially for this sample. The Spin Column can be reused but only for this sample.

2. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection Tube.

3. Add 750µl of Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column.

4. Centrifuge at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough. Reinsert the Spin Column into the Collection Tube.

5. Repeat the wash procedure using 250µl of Column Wash Solution.

6. Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.

7. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at maximum speed.

8. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube.

9. Elute the plasmid DNA by adding 100µl of Nuclease-Free Water to the Spin Column. Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.

10. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.

11. DNA is stable in water without addition of a buffer if stored at –20°C or below. DNA is stable at 4°C in TE buffer. To store the DNA in TE buffer, add 11µl of 10X TE buffer to the 100µl of eluted DNA. Do not add TE buffer if the DNA is to be used for automated fluorescent sequencing.

12. Cap the microcentrifuge tube and store the purified plasmid DNA at –20°C or below.

Vacuum Protocol
Attach one Vacuum Adapter with Luer-Lok® fitting to one port of the manifold (e.g., a Vac-Man® Laboratory Vacuum Manifold). Insert a Spin Column into the Vacuum Adapter until snugly in place.

1. Transfer the cleared lysate (approximately 850µl, Section 3.B, Step 6) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant.
   Note: If the white precipitate is accidentally transferred to the Spin Column, pour the Spin Column contents back into a sterile 1.5ml microcentrifuge tube and centrifuge for another 5–10 minutes at maximum speed. Transfer the resulting supernatant into the same Spin Column that was used initially for this sample. The Spin Column can be reused but only for this sample.

2. Apply a vacuum of at least 15 inches of mercury (Hg) to pull the liquid through the Spin Column. When all liquid has been pulled through the column, release the vacuum.
   **Table Comparing Inches of Hg to Other Pressure Measurements

**

3. Add 750µl of the Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column.
4. Apply a vacuum to pull the Column Wash Solution through the Spin Column. When all the liquid has been pulled through the Spin Column, release the vacuum.
5. Repeat the wash procedure using 250µl of Column Wash Solution. Apply a vacuum to pull the liquid through the Spin Column.
6. Dry the Spin Column by applying a vacuum for 10 minutes.
7. Turn off the vacuum and transfer the Spin Column to a 2ml Collection Tube. Centrifuge at maximum speed for 2 minutes to remove any residual Column Wash Solution. Discard the 2ml Collection Tube and any liquid collected during this step.
8. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube.
9. Elute the plasmid DNA by adding 100µl of Nuclease-Free Water to the Spin Column. Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.
10. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.
11. DNA is stable in water without addition of a buffer if stored at –20°C or below. DNA is stable at 4°C in TE buffer. To store the DNA in TE buffer, add 11µl of 10X TE buffer to the 100µl of eluted DNA. Do not add TE buffer if the DNA is to be used for automated fluorescent sequencing.
12. Cap the microcentrifuge tube and store the purified plasmid DNA at –20°C or below.

Supplementary Information

Selection and Preparation of Plasmids and E. coli Strains
Plasmid DNA can be purified from overnight cultures of E. coli with the Wizard® Plus SV Minipreps DNA Purification System. The yield of plasmid will vary depending on a number of factors, including the plasmid copy number, cell density of bacterial culture, type of culture medium and bacterial strain used. Plasmid copy number is an important factor affecting DNA yield. Copy number is determined primarily by the region of DNA surrounding and including the origin of replication. This region, known as the replicon, controls replication of plasmid DNA by bacterial enzyme complexes. Some DNA sequences, when inserted into a particular plasmid, can lower the copy number of the plasmid by interfering with replication. Choose a single, well-isolated colony from a fresh Luria-Bertani (LB) agar plate (containing antibiotic) and use the colony to inoculate 1–10ml of LB media (also containing antibiotic). The inoculated medium should be incubated overnight (12–16 hours) at 37°C. An A600 of 2.0–4.0 for high-copy-number plasmids ensures that bacteria have reached the proper growth density for harvesting and plasmid DNA isolation.

Choosing a Bacterial Strain
Endonuclease I is a 12kDa periplasmic protein that degrades double-stranded DNA. This protein is encoded by the gene endA. The E. coli genotype endA1 refers to a mutation in the wildtype endA gene, which produces an inactive form of the nuclease. E. coli strains with this mutation are referred to as EndA–. Table 1 contains a list of EndA– and EndA+ E. coli strains. The absence of endA1 (or endA) in an E. coli genotype denotes the presence of the wildtype gene, which expresses an active endonuclease I. The wildtype is indicated as EndA+. Using the Wizard® Plus SV Minipreps DNA Purification System, high- quality DNA is easily obtained from both EndA+ and EndA– strains. However, some EndA+ strains can be problematic for a number of applications. In general, we recommend the use of EndA– strains whenever possible, particularly for applications such as automated fluorescent sequencing. For applications such as fluorescent DNA sequencing, special considerations should be given to the selection of plasmid and E. coli strains to optimize yield and plasmid quality. Optimal automated fluorescent sequencing results are obtained by using high-copy-number plasmids and EndA– strains of E. coli for plasmid propagation.

Table 1. EndA– and EndA+ Strains of E. coli.

Use of Alkaline Protease
To improve the quality of plasmid DNA isolated from both EndA+ and EndA– strains of E. coli, the Wizard® Plus SV Minipreps DNA Purification System includes an alkaline protease solution. Alkaline protease, originally identified as subtilisin Carlsberg, is isolated from the bacterium Bacillus licheniformis (1). Approximately 250µg are added per sample at the end of the lysis step during the preparation of a cleared bacterial lysate to inactivate endonucleases. The alkaline protease also acts to nonspecifically degrade proteins, thus reducing the overall level of protein contaminants in the cleared bacterial lysate (2,3). Alkaline protease is useful in this procedure, because it is optimally active at pH 9 and above, the conditions present during the alkaline lysis procedure. When the lysate is neutralized, alkaline protease activity is substantially reduced (4,5). The DNA prepared by this procedure has been tested extensively in a range of molecular biology applications including fluorescent sequencing, restriction enzyme digestion and cloning.

Considerations for Automated Fluorescent Sequencing
For the application of automated fluorescent sequencing, special consideration should be given to the selection of plasmid type and E. coli strain to optimize yield and plasmid quality.
Note: Optimal automated fluorescent sequencing results are routinely obtained by using high-copy-number plasmids and EndA– strains of E. coli. Purified plasmid DNA must be within the proper concentration range for successful automated cycle sequencing (ideally 0.2µg/µl and not less than 0.1µg/µl). When working with DNA from low-copy-number plasmids, we strongly recommend that DNA concentration be determined by agarose gel/ethidium bromide quantitation prior to any application (6). DNA quantitation by spectrophotometric methods is prone to errors and requires a large amount of sample. The Wizard® Plus SV Minipreps System routinely yields 3.5–5.0µg of plasmid DNA when using a pGEM® Vector and DH5α™ cells in 1.5ml of LB medium. For low-copy-number plasmids, a larger culture volume is required to obtain sufficient DNA for sequencing. Typical low-copy-number plasmid yields are 1.5–3.0µg of plasmid DNA from 10ml of LB culture medium using the pALTER®-1 Vector and DH5α™ cells.

Special Considerations for Sequencing Using BigDye™ Chemistry
The Wizard® Plus SV Minipreps System yields template suitable for use in a number of fluorescent dye sequencing methods, including BigDye™ terminator reactions (Applied Biosystems).
When performing dilutions of the BigDye™ terminator-ready reaction mix, it is essential to dilute the reaction mix using the appropriate dilution buffer (250mM Tris-HCl [pH 9.0], 10mM MgCl2).
When using high-copy-number plasmids, over 500 bases of readable sequence can be obtained from terminator-ready reaction mixes diluted as much as sixfold.
Table 2 outlines the amount of terminator-ready reaction mix and dilution buffer required to obtain the appropriate dilution for BigDye™ terminator reactions. For details on running these reactions, please refer to the protocol supplied with the BigDye™ terminator system. For each reaction, add the reagents in Table 2 to a separate tube.

Table 2. Appropriate Dilutions for BigDye™ Terminator Reactions.

Troubleshooting

For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at: www.promega.com. Email: techserv@promega.com

Composition of Buffers and Solutions

• 5X dilution buffer
  • 250mM Tris-HCl (pH 9.0)
  • 10mM MgCl2
• 10X TE buffer
  • 100mM Tris-HCl (pH 7.5)
  • 10mM EDTA
• LB medium
  • 10g casein peptone
  • 5g yeast extract
  • 5g NaCl
  • 15g agar (for plates only)
  • Dissolve in 1L of distilled water. Autoclave and cool to 55°C before adding antibiotic.
• Cell Lysis Solution (CLA)
  • 0.2M NaOH
  • 1% SDS
• Cell Resuspension Solution (CRA)
  • 50mM Tris-HCl (pH 7.5)
  • 10mM EDTA
  • 100μg/ml RNase A
• Neutralization Solution (NSB)
  • 4.09M guanidine hydrochloride
  • 0.759M potassium acetate
  • 2.12M glacial acetic acid
  • Final pH is approximately 4.2.
• Column Wash Solution (CWA)
  • 162.8mM potassium acetate
  • 22.6mM Tris-HCl (pH 7.5)
  • 0.109mM EDTA (pH 8.0)
    Add 35ml of 95% ethanol for the 50-prep system (170ml for the 250-prep system, 7ml for the 10-prep system) as described in Section 3. Final concentrations will be approximately 60% ethanol, 60mM potassium acetate, 8.3mM Tris-HCl, 0.04mM EDTA.

References

1. Guntelberg, A.V. and Otteson, M. (1954) Purification of the proteolytic enzyme from Bacillus subtilis. Compt. Rend.
   Trav. Lab. Carlsberg 29, 36–48.

2. Aehle, W. et al. (1993) Rational protein engineering and industrial application: Structure prediction by homology
   and rational design of protein-variants with improved ‘washing performance’—the alkaline protease from Bacillus
   alcalophilus. J. Biotechnol. 28, 31–40.

3. von der Osten, C. et al. (1993) Protein engineering of subtilisins to improve stability in detergent formulations.
   J. Biotechnol. 28, 55–68.

4. Vetter, R. et al. (1994) Highly alkaline proteases. U.S. Pat. No. 5,352,603. (October 4, 1994).

5. Shetty, J.K., Patel, C.P. and Nicholson, M.A. (1995) Method of preparation of purified alkaline protease. U.S. Pat. No. 5,439,817. (August 8, 1995).

6. Kahn, M. et al. (1979) Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Meth. Enzymol. 68, 268–80.

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Summary of Changes

The following changes were made to the 4/24 revision of this document:

1. Cover image and font were updated.
2. Expired legal disclaimers were removed.
3. Third party trademarks were updated.
4. Cat.# A1465 was added to Section 2.

© 2010, 2024 Promega Corporation. All Rights Reserved.
pALTER, pGEM, RNasin, Vac-Man and Wizard are registered trademarks of Promega Corporation. PureYield is a trademark of Promega Corporation.
BigDye is a trademark of Thermo Fisher Scientific. DH5a is a trademark of Life Technologies, Inc. Luer-Lok is a registered trademark of Becton, Dickinson and Company.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our website for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.

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