Pulsed-field Gel Electrophoresis Typing of Gram-negative Bacteria (E.coli)

Materials and Reagents

1. Chromosomal low melting agarose (Lonza InCert Agarose or Bio-Rad Laboratories)
2. Restriction enzymes (New England Biolabs)
3. Proteinase K (Promega Corporation) 
4. 10x Tris-borate EDTA (TBE) buffer (Promega Corporation)
5. Agarose for pulsed field gel (Bio-Rad Laboratories) 
6. Lamda ladder for pulsed field gel (Bio-Rad Laboratories)
7. Ethidium bromide (10 mg/ml) (Life Technologies, Invitrogen™)
8. Agar media
9. TE
10. NaCl
11. EDTA
12. Sarkosy l
    
13. Sma I
    
14. Xba I
15. Restriction enzyme buffer
16. Running buffer (0.5x TBE)
17. Suspension buffer (see Recipes)
18. Lysis buffer (see Recipes)
    

Equipment

1. CHEF-Mapper^TM apparatus (Bio-Rad Laboratories)
2. Spectrophotometer (Bio-Rad Laboratories) 
3. UV light and photograph
4. Water bath
5. Glass rod
6. 1.5 ml Eppendorf tube
   

Procedure