recombinant DNA technology

Experiment 4

Aim of the Experiment

Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Introduction

Plasmid is an extra-chromosomal DNA molecule different from the chromosomal DNA which is capable of independent replication. The term “Plasmid” was first introduced by the American molecular biologist Joshua Lederberg in 1952. Plasmid size varies from 1 to over 1000 kilo base pairs (kbps). Plasmids are mostly circular and double-stranded. Plasmids are found in a wide variety of bacterial species; but are sometimes found in eukaryotic organisms e.g., in Saccharomyces cerevisiae.

Plasmids typically have two important elements:

Ø An origin of replication

Ø A selectable marker gene (e.g. resistance to ampicillin)

Conformations of plasmid: Plasmid DNA may appear in one of the five conformations which are as follows:

Ø “Nicked Open-Circular” DNA has one strand cut.

Ø “Relaxed Circular” DNA is fully intact with both strands uncut, but has been enzymatically “relaxed” (supercoils removed).

Ø “Linear” DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo.

Ø “Supercoiled” (or “Covalently Closed-Circular”) DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.

Ø “Supercoiled Denatured” DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact.

The conformations listed above are in order of electrophoretic mobility from slowest to fastest; and for a given size, run at different speeds in the gel during electrophoresis.

Principle

The alkaline lysis method for isolation of plasmid DNA from E.coli has been used for more than 20 years. This method takes advantage of the physical difference between linear, closed and supercoiled DNA. The bacterial suspension is first exposed to a strong anionic detergent (i.e. SDS) at high pH which helps to rupture the cell wall by hyper-lytic osmosis releasing the DNA (chromosomal and plasmid), proteins and other contents which are denatured. The strands of the closed circular plasmid DNA are not completely ruptured in this process as the plasmid has a highly supercoiled confirmation. On addition of neutralization solution, the proteins, polysaccharides and genomic DNA are precipitated, whereas the plasmid DNA remains in the solution. The plasmid DNA is then precipitated by addition of isopropanol. Subsequently, other contaminants are removed by addition of Wash Solution I and II. The pure plasmid is then eluted in Elution Buffer.

Materials required

1. RNAase

2. Isopropanol

3. Ethanol Absolute

4. Tryptone

5. Yeast extract

6. NaCl

7. Glucose

8. EDTA

9. 0.2 N NaOH

10. SDS

11. KOAc

12. Potassium acetate

13. Glacial acetate

14. Tris-HCl (pH 8.0)

15. Luria-Bertani broth (LB) medium: Bacto-tryptone, yeast extract.

16. Resuspension solution (P1 buffer)

17. Lysis solution (P2 buffer)

18. Neutralizing solution (P3 buffer)

19. TE

• How to prepare buffers:

1. Resuspension solution (P1 buffer)
   50 mM glucose
   10 mM EDTA
   25 mM Tris (pH 8.0)
   Store at 40 °C

2. Lysis solution (P2 buffer)
   0.2 N NaOH
   1% SDS
   Store at room temperature

3. Neutralizing solution (P3 buffer)
   3 M KOAc (pH 6.0)
   For 100 ml solution, 60 ml 5 M potassium acetate (49.07 g potassium acetate in 100 ml H₂O)
   11.5 ml glacial acetate and 28.5 ml H₂O, store at room temperature.

4. TE
   1 mM EDTA
   10 mM Tris-HCl (pH 8.0)

Procedure

• Day 1: Revival of Host

1. Open the vial containing culture and resuspend the cells with 0.25 ml of LB broth.

2. Pick up a loopful of culture and streak onto LB agar plate with ampicillin

3. Incubate overnight at 37°C.

• Day 2: Inoculation of culture

1. Pick up a single colony from LB agar plate and inoculate in 10 ml of LB broth containing 10 μl ampicillin.

2. Incubate the test tube overnight at 37°C.

• Day 3: Plasmid Extraction

1. Take 1.5 ml of the overnight grown culture into a micro centrifuge tube and centrifuge the cells at 13,000 rpm for 3 minutes. Discard the supernatant culture medium.

NOTE: For good plasmid yields, the O.D 600 of the culture should be around 3.0 x 10 ⁶ cells/ml.

2. Resuspend the cell pellet in 250 μl of Resuspension Solution and mix well by gentle vortexing till no cell clumps are visible .

3. Add 250 μl of Lysis Solution to lyse the cells. Mix thoroughly by gently inverting the tube 4-6 times.

NOTE: Do not vortex the tubes as it may result in the shearing of genomic DNA which may contaminate the plasmid DNA. Do not allow this lysis reaction to exceed for more than 5 minutes.

4. Add 350 μl of Neutralization Solution and immediately mix thoroughly by inverting the tube gently 4- 6 times.

NOTE: On addition of Neutralization Solution, genomic DNA will precipitate out. The mixture should become cloudy and the precipitation should be homogeneous.

5. Centrifuge the sample at 13,000 rpm for 10 minutes to obtain a compact white pellet.

NOTE: A compact white pellet of genomic DNA will form. If the supernatant is not clear, transfer the supernatant to a fresh tube and spin for an additional one minute at 13,000 rpm to remove the interfering salts/precipitates completely.

6. Precipitation of Plasmid DNA- Carefully transfer the supernatant containing plasmid DNA to a new collection tube. Add 1 ml of isopropanol to precipitate plasmid DNA and mix by gentle inversion for 5 minutes.

7. Centrifuge at 13,000 rpm for 15 minutes. White pellet of plasmid DNA will be seen, sticking to the sides of the tube. Discard the supernatant and invert the vial on blotting paper to drain out left over supernatant.

8. Resuspend the pellet by adding 500 μl of Wash Solution I (HPB) and centrifuge at 13,000 rpm for 3 minutes.

9. Discard the supernatant and air dry the pellet for 10-15 minutes at room temperature.

10. DNA Elution - Resuspend the pellet in 50 μl of Elution Buffer. Centrifuge at 13,000 rpm for 5 minutes to remove insoluble material and transfer the supernatant containing pure plasmid DNA into a new collection tube.

11. Storage of the eluate with purified DNA: The eluate contains pure plasmid DNA. For short term storage (24-48 hours) of the DNA, 2-8°C recommended. For long-term storage, -20°C or lower temperature (-80°C) is recommended. Avoid repeated freezing and thawing of the sample which may cause denaturation of DNA. The Elution Buffer will help to stabilize the DNA at these temperatures.

• Agarose Gel Electrophoresis:

Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.

Preparation of agarose gel: To prepare 50 ml of 0.8% agarose gel, add 0.4 g agarose to 50 ml 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plate or burner by swirling the glass

beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60oC. Add 0.5 μl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

NOTE: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.

Loading of the DNA samples: To prepare sample for electrophoresis, add 2 μl of 6X gel loading buffer to 10 μl of DNA sample. Mix well by pipetting and load the sample into the well. Load the Control DNA after extracting the DNA sample.

Electrophoresis: Connect power cord to the electrophoretic power supply according to the conventions: Red- Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

* Molecular biology grade water is recommended.

Observation & Result

Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator and calculate the yield and purity using UV Spectrophotometer.

Reference Material

T.A. Brown- Gene Cloning Book.

https://bio-protocol.org/bio101/e30

Questions

1. How one can interpret which type of Plasmid can run faster?

2. What is ladder and its purpose?

3. Size of the plasmid?

4. Why EtBr is used to visualized DNA? Is there any other agent?

5. In which direction does DNA run in the electrophoretic unit, cathode to anode or vice-versa? Justify your answer.

6. In what ways does Isopropanol different from ethanol in DNA separation?