Isolation of Plasmids

Introduction
Plasmids are extrachromosomal double-stranded closed-circular DNA present in many microorganisms. Plasmids are usually present in the cell conferring extraordinary properties to the cell, like the ability to conjugate, conferring antibiotic resistance, degradation of xenobiotic substances, production of substances to neutralize toxins, etc.

In nature, plasmids are large molecules with sizes ranging from 2 Kb to 100 Kb. Plasmids have origin of replication and multiply by utilizing the longleaved enzymes of the host DNA polymerizes. However, the plasmids used in molecular cloning are different from the natural plasmids, as they are small in size and offer multiple restriction sites.

Plasmid DNA needs to be extracted (from bacterial hosts, mostly E.coli) almost routinely in cloning experiments. Many methods have been described for successful extraction of plasmid DNA; however, they can be grouped into 2 categories:

1. Miniprep method and
2. Two methods have been described here representing both the categories. The alkaline miniprep method is most useful for quick extraction of plasmids, mostly for analytical use, whereas, the large-scale preparation, is most useful for preparative extraction.

Alkaline Miniprep Method
The principle of alkaline miniprep method involves the lysis of bacterial cells followed by SDS, NaOH treatment. The high pH of NaOH denatures the bacterial DNA but not the covalently closed-circular plasmid DNA. Neutralization of the high pH by sodium or potassium acetate makes the bacterial DNA to precipitate. The plasmid DNA is then purified by organic solvent.

Growth of Bacteria and Amplification of Plasmids
Plasmid DNA can be isolated from bacterial culture, which is grown in a liquid medium containing appropriate antibiotic. The bacterial culture should be grown in LB medium, inoculated with a single colony picked from an agar plate. For low-copy plasmids like pBR322, chloramphenicol is to be added after the culture attains late-log phase (A₆₀₀=0.6) and shaken vigorously for several hours to amplify the plasmid. However, for very high-copy-number plasmids like pUC-series plasmids, such amplification is not required.

Materials

• L-Broth: Trypton, 1%; Yeast extract, 0.5% NaCl, 1%
• Antibiotics: Ampicillin, 50 mg/mL
• Solution I: 50 mM glucose, 25 mM Tris, 10 mM EOT A, pH 8.0
• Solution II: 0.2 M NaOH, 1% SDS
• Solution III: 5M Potassium acetate, 60 mL; acetic acid, 11.5 mL; distilled water, 28.5 mL.
• Phenol: Chloroform
• Chloroform: isoamyl alcohol (24:1)
• TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0
• 70% and 100% ethanol.

Harvesting and Lysis of Bacteria
Harvesting: Transfer a single bacterial colony into 2 mL of LB medium containing the appropriate antibiotic in a loosely capped 15-ml tube.

Incubate the culture overnight at 37°C with vigorous shaking. Pour 1.5 mL of the culture into a centrifuge tube. Centrifuge at 12000 g of 5 seconds at 4°C a microphage.

Store the remainder of the culture at 4°C. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.


Lysis by Alkali
Resuspend the bacterial pellet in 100 mL of ice-cold Solution I by vigorous vortexing.

Solution I
- 50 mM glucose
- 25 mM Tris-CI (pH 8.0)
- 10 mM EDT A (pH 8.0)
→ Solution I can be prepared in batches of approximately 100 mL autoclaved for 15 minutes at 10 lb/sq and stored at 4°C.
→ Add 200 mL of freshly prepared Solution II.

Solution II
- 0.2 N NaOH (freshly diluted from 10 N stock)
- 1% SDS
→ Close the tube tightly and mix the contents by inverting the tube rapidly for 5 minutes. Make sure that the entire surface of the tube comes in contact with Solution II. Do not vortex. Store the tube on ice.
→ Add 150 mL of ice-cold Solution III.

Solution III

- 5 M potassium acetate	60 mL
- Glacial acetic acid	11.5 mL
- Water	28.5 mL

The resulting solution is 3 M with respect to potassium Clod 5 M with respect to acetate.

Close the tube and vortex it gently in an inverted position for 10 seconds to dispers

Solution III through the viscous bacterial lysate. Store the tube on ice for 3–5 minutes.

Centrifuge at 12 xg for 10 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube.

Add an equal volume of phenol:chloroform. Mix by vortexing. After centrifuging at 12 xg for 10 minutes at 4°C in a microfuge, transfer the supernatant to a fresh tube.

• Precipitate the double-stranded DNA with 2 volumes of ethanol at room temerature. Mix by vortexing. Allow the mixture to stand for 2 minutes at room temperature.
• Centrifuge at 12 xg for 10 minutes at 4°C in a microfuge.
• Remove the supernatant by gentle aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube.
• Rinse the pellet of double-stranded DNA with 1 mL of 70% ethanol at 4°C. Remove the supernatant as described in previous step, and allow the pellet of nucleic acid to dry in the air for 1 minute.
• Redissolve the nucleic acids in 50 mL of TE (pH 8.0) containing DNAsefree RNAse 20 mg/mL. Vortex briefly. Store the DNA at –20°C.

Important Notes

• The original protocol requires the addition of lysozyme. Before the addition of solution II, this is not necessary.
• Do not vortex the tubes after addition of solution II.
• If the plasmid preparation is strictly for analytical purposes and nonenzymatic manipulations are contemplated, then the phenol:chloroform step can be avoided.
• It is important to remove all the supernatant fluid after harvesting the bacterial pellet and all traces of ethanol, etc., after precipitation.
• The phenol has to be tris-saturated to pH 8.0 and of very good quality.
• Do not disturb the whitish interface while removing the upper aqueous phase after phenol and chloroform treatments.
• While washing with 70% ethanol, do not break the DNA pellet. This step is meant for washing the pellet only to remove traces of ethanol and salts. If the pellet is disturbed at this stage, it will be difficult to recover the DNA.
• This preparation will contain a lot of RNA contamination. DNAse-free RNAse may be added before the phenol:chloroform step to digest the RNA.
  Otherwise RNAse may be added, along with the restriction enzyme, during subsequent manipulations.
• The protocol may be upgraded to accommodate up to 10 mL of bacterial culture.
• Sometimes it may become difficult to dissolve the plasmid preparation in IE. Keep it in the freezer overnight. The next day, the DNA will easily dissolve.