Plasmid DNA Isolation Protocol

Plasmid DNA Isolation Protocol

Aim: To isolate pUC-19 plasmid from E.coli cells.

Principle:

Bacterial plasmids are self replicating, circular extra chromosomal DNA molecules. The most convenient method for preparing plasmid DNA is the alkali lysis method of BIRNBOIMS AND DOLY (1979). Alkaline lysis is the method of choice for isolating circular plasmid DNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.  Alkaline lysis depends on a unique property of plasmid DNA. It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.

Typically, we grow up E coli cells that contain the plasmid we want to isolate, then we will lyse the cells with alkali and extract the plasmid DNA. The cell debris is precipitated using SDS and potassium acetate. This is spun down, and the pellet is removed. Isopropanol is then used to precipitate the DNA from the supernatant, the supernatant is removed, and the DNA is re-suspended in buffer (often TE).

Chemicals used:

• STE buffer – to remove any media remaining in the bacterial pellet.
• Glucose – Prevents immediate lysis of spheroplast.
• EDTA – Chelates metal ions required for nuclease activity.
• Tris – maintains pH
• NaOH – Lysis of the cells
• SDS – Denatures plasmid and chromosomal DNA.
•  Phenol: chloroform: isoamyl alcohol – remove protein concentration
• Chloroform: isoamyl alcohol – removes traces of phenol in the supernatant
•  70% ethanol – To remove excess salt and other water soluble impurities
• Acetic acid - To decrease the pH.

Requirements:

Culture: Colonies of E.coli cells containing pUC19 or pGPTV plasmid.

Media : Leuria Bertini broth

    Tryptone: 10 gm

    Yeast extract: 10 gm

    NaCl: 5 gm

    Distilled water: 1000 ml

Ready-to-use LB broth powder: Dissolve 20gms in 1000ml Distilled water.

Agar: 15g in 1000ml distilled water

Reagents required:

STE solution

     0.1 M NaCl

     10 mM Tris-HCl (pH 8.0)

     1mM EDTA [pH 8.0]

Solution 1

      50 mM Glucose

      25 mM Tris-HCl (pH 8.0)

      10 mM EDTA (pH 8.0)

Solution 2 [Freshly prepared]

     0.2 N NaOH

     1% SDS (freshly prepared)

Solution 3

     5M potassium acetate – 60ml

     Glacial acetic acid – 11.5 ml

     Double distilled water – 28.5 ml

Equilibrated phenol: Phenol equilibration: Before use phenol was equilibrated to pH 8.0 because DNA will partition into organic phase at this pH.

1. Liquefied phenol was stored at -20º C. As needed phenol was removed  From the freezer and allowed to warm to room temperature and then melted to 68º C.  0.1% Hydroxyquinoline was added to the phenol. This compound is an Antioxidant, a partial inhibitor of RNase and a weak chelator of metal ions.
2. To the melted phenol an equal volume of 0.5M Tris-HCl buffer {pH 8}was added at room temperature. The mixture was stirred for 15 min on a stirrer and was switched off when 2 phases had separated. The upper phase was removed using a glass pipette.
3. Equal volume of 0.1M Tris-HCl{pH 8} was added to phenol, the mixture  was then stirred on a magnetic stirrer for 15 min. and was switched off. The upper phase was removed. This step was repeated 3 times until the pH of the phase is > 7.8.
4. After phenol was equilibrated and the final aqueous phase had been removed 0.1 volume of 0.1M Tris-HCl containing 0.2% B-mercaptoethanol was added. The phenol solution was stored in this form under 100mM Tris-HCl in a brown bottle at 4º C for a period of up to one month.     

Mixture of equilibrated phenol: chloroform: isoamyl alcohol (25:24:1)

Chloroform: isoamyl alcohol (24:1)

    

100% ethanol and 70% ethanol

               

Tris EDTA buffer (pH 8.0)

                100mM Tris-HCl (pH 8.0)

                1 mM EDTA (pH 8.0)

  

3M sodium acetate (pH 5.2)

                 Dissolve 408.1 gm of CH₃COONa in 800ml of water and adjust the pH to 5.2

                 with glacial acetic acid. Make-up the volume to 1L with water and sterilize

                 by  autoclaving.

Antibiotics

Kanamycin (100mg/ml)For pGPTV

                  Kanamycin – 100mg

                  Double distilled water – 1 ml.

Ampicillin (100mg/ml)For pUC19

           Ampicillin – 100mg

          Double distilled water – 1ml

Procedure: Plasmid isolation involves 4 steps:

1. Harvesting: Grow cultures overnight and centrifuge to pellet down the cells  and resuspend the pellet in STE and again recover by centrifugation.
2. Alkali lysis: Treat the pellet with solution 1, 2 and 3 whose constituents help to lyse the cells and precipitate the cell debri and protein content by centrifugation.
3. Plasmid precipitation: Treat the supernatant with 0.6 volume of Isopropanol in order to selectively precipitate only plasmid DNA. Recover DNA by centrifugation.
4. Purification: Dissolve the pellet in TE buffer and treate with RNase 'A'. Further apply phenol : chloroform extraction to remove protein and cell debris. Precipitate the purified DNA 0.1 v of 3M sodium acetate and 100% ethanol, finally wash with 70% ethanol, dried & stored at  -20°C.