MiniPrep

3.B. 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 centrifugation for 5 minutes at 10,000 x 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

proceeding to addition of 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.

3.C. 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.

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.