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


  • 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 can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

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


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


  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A


Preparation of 100 ml of resuspension buffer (solution I)


Step 1: To prepare 100 ml of resuspension buffer, take 94.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.


The solution can be stored at 4°C for 3 – 6 months.

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


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
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 94.5 ml