Preparation of Resuspension Buffer Containing Tris and EDTA for Isolation of Plasmid by Alkaline Lysis Method

Overview:

  • The resuspension buffer is used to resuspend bacterial cells during plasmid isolation by the 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 the 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

To prepare 100 ml of resuspension buffer, take 95.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker. 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 the resuspension buffer.

Precaution:
Do not mix concentrated stock solutions. This can cause precipitation.

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

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

Application
Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare the resuspension buffer of various volumes.

Reagents / Volume10 ml25 ml50 ml100 ml
1M Tris.Cl (pH 8.0)0.25 ml0.625 ml1.25 ml2.5 ml
0.5 M EDTA (pH 8.0)0.2 ml0.5 ml1.0 ml2.0 ml
Water9.45 ml23.625 ml47.25 ml95.5 ml

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