Yeast DNA Replication 2D Gel Protocol

Materials and Reagents

1. Qiagen genomic-tip 20/G ( QIAGEN, catalog number: 10223 , if more DNA needed, 100/G could be used to isolate DNA)
   
2. Qiagen Buffer G2 ( QIAGEN, catalog number: 1014636 )
   
3. Qiagen buffer QBT (QIAGEN, catalog number: 19054 )
   
4. Qiagen buffer QC (QIAGEN, catalog number: 19055 )
   
5. Qiagen buffer QF ( QIAGEN, catalog number: 19056 )
   Tips: Buffer QBT, QC and QF could be made by yourself.
   
6. RNase A (10 mg/ml) ( QIAGEN, catalog number: 19103 )
   
7. Proteinase K (20 mg/ml) (QIAGEN, catalog number: 19131 )
   Tips: RNase A and Proteinase K could be ordered from any other company, they work fine too.
   
8. Sodium azide
   
9. Isopropanol
   
10. Tris-HCl
    
11. EDTA
    
12. Agarose
    
13. KoAc
    
14. Ethidium bromide
    
15. Glycerol
    
16. MgCl₂
    
17. Spermine
    
18. Spermidine
    
19. KOH
    
20. GuHCl
    
21. MOPS free acid
    
22. Tween-20
    
23. Triton X-100
    
24. NaCl
    
25. KB ladder
    
26. 70% ethanol
    
27. TE (see Recipes)
    
28. Nuclear isolation buffer (NIB buffer) (see Recipes)
    
29. Sodium azide solution (see Recipes)
    
30. Qiagen Buffer QBT (see Recipes)
    
31. Qiagen Buffer QC (see Recipes)
    
32. Qiagen Buffer QF (see Recipes)
    

Equipment

1. Acid-washed glass beads (425-600 μm diameter) (Sigma-Aldrich, catalog number: G8772-500G )
   
2. BioRad sub-cell GT gel box or other Maxi horizontal electrophoresis units (big agarose gels are needed for both dimentions)
   
3. 4 °C cold room
   
4. Micro centrifuges
   
5. Vortex mixer (VORTEX-GENIE)
   
6. Microwave oven
   
7. Hand-held UV lamp
   
8. UV light box
   
9. 2D gel apparatus tray (4 samples per apparatus -- Fischer self-circulating gel box)
   
10. 50 ml polypropylene centrifuge tubes
    
11. 2-ml polypropylene tube
    
12. Pasteur pipette
    
13. 12 x 75 mm polypropylene culture tube
    

Procedure