Preparation of resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer containing Tris and EDTA is very common.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • EDTA  chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Resuspension buffer can be supplemented with RNase A which helps to get rid of RNA contamination from the plasmid preparation.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 95.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 95.5 ml