- Resuspension buffer (solution I) is used for the isolation of plasmid DNA by alkaline lysis method. Bacterial cells, obtained from the culture (liquid culture or colonies, grown on an agar plate), are resuspended in this buffer. The purpose of the resuspension buffer is to provide an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
- The classical composition of the resuspension buffer (designed by Birnboim and Doly) contained Lysozyme, Glucose, Tris.Cl, and CDTA (or EDTA). Most of the recent formulations do not contain lysozyme and glucose.
- Lysozymes are glycoside hydrolases that destroy bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan. However, for most bacteria including E. coli DH5α, lysis solution was found to induce complete lysis, thus eliminating the use of lysozymes.
- Glucose is added to make the solution isotonic. However, isotonicity is not required for the cell wall containing bacteria including E. coli DH5α. The cell wall containing bacteria can withstand a wide range of solution concentrations. Therefore, Glucose is not included in the recent recipes of resuspension buffer. Furthermore, glucose-containing resuspension buffers cannot be stored for a long time, and need to be kept at 4°C. However, if lysozyme is included in the resuspension buffer, glucose must be added as lysozyme action destroys the cell wall, leading to lysis of bacteria if the resuspension buffer is not isotonic.
- EDTA (or CDTA) 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.
- Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
- Now researchers prefer to supplement the resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under very stringent conditions including high alkaline condition, the presence of detergent, and chelating agent (EDTA). The addition of RNase A in the resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria. However, such plasmid preparation cannot be used for in-vitro transcription due to the contamination of RNases. In addition, RNase A containing resuspension buffers should be stored at 4°C and has a limited life (1 month) as RNase A activity diminishes with time in solution.
- pH indicator, LyseBlue from Qiagen, can also be added to the resuspension buffer. LyseBlue ensures the complete lysis and subsequent neutralization step. Both steps are very important to get high-quality plasmid DNA.
- The resuspension buffer is not included in the protocol of plasmid isolation using a plasmid isolation kit provided by some manufacturers (see Zyppy Plasmid Miniprep Kit). In those procedures, a highly concentrated lysis buffer is added directly to the overnight grown liquid culture of bacterial cells.
- The following types of resuspension buffer can be used for plasmid isolation
- Resuspension buffer with glucose: 50 mM Glucose, 25 mM Tris.Cl (pH 8.0), 10 mM EDTA (pH 8.0)
- Resuspension buffer without glucose: 25 mM Tris.Cl (pH 8.0), 10 mM EDTA (pH 8.0), 100 μg/ml RNase A
- Resuspension buffer is prepared without RNase A or lysozyme. Depending on the requirement, RNase A (final concentration of 100 μg/ml) can be added to any of them. Lysozyme (2 mg/ml) can only be added to glucose-containing resuspension buffer.
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