- Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
- Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
- Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.
- 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 the read article: Preparation of resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.
- 1M Glucose solution, filter sterilized
- 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)
- 50 mM Glucose
- 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)
- Do not mix concentrated stock solutions together. This can cause precipitation.
- 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
|Reagents / Volume||10 ml||25 ml||50 ml||100 ml|
|1M Glucose solution||0.5 ml||1.25 ml||2.5 ml||5 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||8.95 ml||22.375 ml||44.75 ml||89.5 ml|