Promoter Orientation of Prokaryotic Phase-variable Genes by PCR

Materials and Reagents

1. Escherichia coli (E. coli) isolate UTI893
   
2. Sterile distilled, deionized water (diH₂O)
   
3. Agarose, molecular biology grade, standard sieve
   
4. Ethidium Bromide, 1 mg/ml in distilled, deionized water (diH₂O) (or other agent to visualize DNA)
   
5. Taq polymerase (1 U/μl) with 10x NH₄ buffer (APEX Bioresearch Products, catalog number: 42-409 )
   
6. 10 mM dNTP mix, PCR grade (Life Technologies, catalog number: 18427-013 )
   
7. Tryptone
   
8. Yeast extract
   
9. NaCl
   
10. Tris base
    
11. Boric acid
    
12. EDTA (pH 8.0)
    
13. SDS
    
14. Glycerol
    
15. Xylene cyanol
    
16. Bromophenol blue
    
17. hyxR phase-specific primers, 100 μM stock solution [Integrated DNA Technologies (IDT)]
    5’ – ACTGATAATAACCAGAGGCTTCTT – 3’
    5’ – CAGTGATTAACTTTCGAACATATTG – 3’
    5’ – GCGAAAGTTAATCACTGGTATGACC – 3’
    
18. Tris-Borate-EDTA (TBE) (10x stock) (see Recipes)
    
19. 10x DNA Loading Dye (see Recipes)
    
20. Luria-Bertani broth (LB) culture medium (Sigma-Aldrich, catalog number: L3022-250G (see Recipes)
    
21. 2% TBE agarose gel with EtBr (see Recipes)
    
22. 20 ml 10x DNA Loading dye⁶ (xylene cyanol/bromophenol blue) (see Recipes)
    

Equipment

1. 37 °C Incubator for bacteria with aeration
   
2. Thermal cycler (Bio-Rad Laboratories)
   
3. Gel electrophoresis system (Owl Separation Systems)
   
4. UV Transilluminator with photo documentation (Bio-Rad Laboratories)
   

Procedure