Isolation of plasmid by alkaline lysis

/Isolation of plasmid by alkaline lysis
Isolation of plasmid by alkaline lysis 2018-05-12T06:20:09+00:00

Isolation of plasmid by alkaline lysis

Isolation of plasmid by alkaline lysis

Introduction

Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. Plasmids almost carry one or more genes responsible for a useful characteristic displayed by the host. Classification of the naturally occurring plasmid is based on the main characteristic encoded by the plasmid. The five main types of plasmids are:

  1. Fertility or F-plasmids – these plasmids carry ‘tra’ genes which promote conjugal transfer of the plasmid.
  2. Resistance or R-plasmid – these plasmids carry genes that confer on the host, some antibiotic resistance.
  3. Col plasmids – these plasmids code for colicins (proteins that kill other bacteria)
  4. Degradative plasmids – these plasmids allow the host molecule to metabolise unusual molecules such as toluene and salicylic acid.
  5. Virulence plasmid – these plasmids confer pathogenicity on the host bacterium.
  6. Isolation of plasmids involves the use of three solutions for the extraction and purification of plasmid. These are:
    • Solution I – contains EDTA which lyses the cells and chelates metal ions, thus weakening the cell wall and inactivating enzymes that digest DNA.
    • Solution II – contains SDS and NaOH. SDS is an anionis detergent, which removes the lipid molecules and hence disrupts the cell membrane. It also helps denature the bacterial proteins. Addition of NaOH increases the pH of the solution (hence the name alkaline lysis) thereby denaturing bacterial chromosomal and plasmid DNA.
    • Solution III – contains potassium acetate which reduces pH and therefore the plasmid DNA renatures due to its small size.
  7. The chromosomal DNA strands and bacterial proteins form a precipitate along with SDS which is removed by centrifugation. The renatured plasmid present in the supernatant solution is concentrated by the addition of isopropanol.

Requirements

Reagents

  1. Alkaline lysis solution I
  2. Alkaline lysis solution II
  3. Alkaline lysis solution III
  4. Ethanol
  5. Phenol: chloroform (1:1, v/v)
  6. TE (pH 8.0) containing 20 μg/ml RNase A
  7. Luria broth

Composition of buffers and solutions

Alkaline Lysis Solution I

  1. 50 mM glucose
  2. 25 mM Tris-Cl (pH 8.0)
  3. 10 mM EDTA (pH 8.0)
  4. Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.

Alkaline Lysis Solution II

  1. 0.2 N NaOH (freshly diluted from a 10 N stock)
  2. 1% (w/v) SDS
  3. Prepare Solution II fresh and use at room temperature.

Alkaline Lysis Solution III

  1. 5 M potassium acetate, 60.0 ml
  2. Glacial acetic acid, 11.5 ml
  3. H2O, 28.5 ml
  4. The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.

EDTA
To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Glycerol
To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biologygrade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-μm filter. Store in 200-ml aliquots at 4°C.

TE

  1. 100 mM Tris-Cl (desired pH)
  2. 10 mM EDTA (pH 8.0)
  3. (10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Store the buffer at room temperature.

Equipments

  1. Laminar air flow
  2. Refrigerated micro centrifuge
  3. Incubator
  4. Refrigerator
  5. Vortex

Procedure

  1. Inoculate 2 ml of rich medium (LB) with a single colony of bacteria. Incubate the culture overnight at 37°C with vigorous shaking.
  2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.
  3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
  4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.
  5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.
  6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.
  7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube.
  8. (Optional) Add an equal volume of chloroform:Isoamyl alcohol. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.
  9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.
  10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.
  11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.
  12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.
  13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.
  14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).
  15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.
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