Materials and Reagents

1. 300 ng/µl plasmid DNA
   We used 7.2 kbp pMK 3 plasmid purified from E. coli JM109 by QIA filterWe used 7.2 kbp pMK3 plasmid purified from E. coli JM109 by QIA filter^TM (Plasmid Midi kit). The high quality plasmid abundant in supercoiled form is required to monitor the reduction of the supercoiled form.
   
2. Proteins to be tested on their DNA protection ability
   a. BSA (Wako Pure Chemical Industries, catalog number: 01317843 )
   b. Lysozyme (Wako Pure Chemical Industries, catalog number: 12202673 )
   
3. Agarose
   
4. Ethidium bromide (EtBr) (Invitrogen, catalog number: 15585011 )
   Caution: Ethidium bromide is toxic and strong mutagen. Use appropriate gloves, safety goggles and lab coat
   
5. Ferrous ammonium sulfate (1.5 mM) (Wako Pure Chemical Industries, catalog number: 01412172 )
   Note: The solution must be prepared just prior to the experiment.
   
6. 200 mM hydrogen peroxide (Wako Pure Chemical Industries, catalog number: 08104215 )
   
7. NaCl (Wako Pure Chemical Industries, catalog number: 19101665 )
   
8. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 08104215)
   
9. Tris (Nacalai tesque, catalog number: 3543421 )
   
10. Acetic acid (Nacalai tesque, catalog number: 0021243 )
    
11. EDTA (Nacalai tesque, catalog number: 15111 )
    
12. CIA (chloroform:isoamyl alcohol = 24:1) (Nacalai tesque, catalog number: 0840255 )
    Caution: Chloroform is suspect mutagen and harmful if inhaled. Avoid breathing vapor and prolonged contact with skin, using fume food and safety gloves. Follow the safety rule in your institute.
    
13. 300 ng/µl plasmid DNA (pMK3) (see Recipes)
    
14. 1 µg/µl bovine serum albumin (BSA) (see Recipes)
    
15. 1 µg/µl lysozyme (see Recipes)
    
16. Protein/binding buffer (see Recipes)
    
17. 1.5 mM ferrous ammonium sulfate (see Recipes)
    
18. 200 mM hydrogen peroxide (see Recipes)
    
19. 10% sodium dodecyl sulfate (SDS) (see Recipes)
    
20. 1x TAE buffer (see Recipes)
    
21. 50x TAE buffer (see Recipes)
    

Equipment

1. Eppendorf tubes (1.5 ml)
   
2. Eppendorf tubes (2.0 ml)
   
3. Tips
   
4. Thermostat bath
   
5. Centrifuge machine
   
6. Electrophoresis apparatus
   
7. UV Trans illuminator and recording system (Nippon Genetics, model: e.g. FAS III system )
   

Software

1. Image J (NIH) (http://imagej.nih.gov/ij/)

Procedure