Overview:
- Resuspension buffer is used to resuspend the harvested bacterial cells from the culture during plasmid isolation by the alkaline lysis method.
- 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. 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.
- The glucose-containing resuspension buffer is prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.
To know more, please read the article: Resuspension Buffer (Solution I) for Isolation of Plasmid by Alkaline Lysis Method
Requirements
Reagents and solutions
> 1M Glucose solution, filter-sterilized
> 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
> 50 mM Glucose
> 25 mM Tris.Cl (pH 8.0)
> 10 mM EDTA (pH 8.0)
Objective:
Preparation of 100 ml of resuspension buffer (solution I) containing glucose
Preparation
Step 1: To prepare 100 ml of resuspension buffer, take 90.5 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 5 ml of 1 M glucose solution, 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.
Storage
The solution can be stored at 4°C for 3 months.
Precaution:
Frequently check the presence of any microbial growth in the resuspension buffer. Discard if you detect any microbial growth.
Application:
Plasmid isolation from E.coli by alkaline lysis method
| Follow the table to prepare a resuspension buffer of various volumes. | ||||
| 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 |
| Water | 9.05 ml | 22.625 ml | 45.25 ml | 90.5 ml |
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