Promega Wizard Midiprep Buffers

1. Overview

For routine cloning and plasmid preparations, we use the traditional alkaline-lysis method. When large quantities of high-quality plasmids need to be made, we use the Promega Wizard Midiprep Kit for high-purity plasmid preparation. The columns and resin are purchased from Promega, while the buffers are made in-house.

Midi-prep manuals:

• Quick Protocol
• Full Manual

2. Recipes

When making the buffers, make sure to write your initials and the date on the appropriate buffer bottle. If a buffer is running low, make more for the lab!

2.1 Cell Resuspension Buffer

To make 500 mL of buffer:

Component	Volume to add	Final Concentration
Tris-Cl pH 7.5 (1M)	25 ml	50 mM
EDTA pH 8 (0.5M)	10 ml	10 mM
RNAse A (solid)	50 mg	100 µg/ml
H2O	to 500 ml

2.2 Cell Lysis Buffer

To make 500 mL of buffer: NaOH Molecular Weight: 40 g/mol

Component	Volume to add	Final Concentration
NaOH (solid)	4 g	0.2 M
SDS (solid)	5 g	1 %
H2O	to 500 ml

2.3 Neutralization Buffer

To make 500 mL of buffer: Potassium Acetate Molecular Weight: 98.15 g/mol. Adding glacial acetic acid will solidify your potassium acetate mixture. Add water to place it back in solution.

Component	Volume to add	Final Concentration
Potassium Acetate (solid)	64.8 g	1.32 M

1. Dissolve 64.8 g Potassium Acetate in ~30 ml H2O.
2. Add glacial acetic acid to bring to pH 4.8 (~ 100 mL acetic acid).
3. Bring up to 500 mL with H2O.

2.4 Column Wash Buffer

To make 4 L of buffer: Dissolve everything in water and add ethanol at the end. If using 95% ethanol, adjust water concentration accordingly to get a final ethanol concentration of 55% v/v.

Component	Volume to add	Final Concentration
Potassium Acetate (solid)	31.4 g	80 mM
Tris-Cl pH 7.5 (1 M)	33 ml	8.3 mM
EDTA (0.5 M)	0.32 ml	40 µM
H2O	1.76 L
Ethanol (100%)	2.2 L	55%