Knight:Centrifuge desalting/Zeba columns

Overview

This protocol is for doing buffer exchange on small sample volumes (30-130 μL) volumes.

Materials

• Zeba 0.5mL desalting spin columns from Pierce
• Variable-speed bench-top microcentrifuge
• 2.0 mL collection tubes
• Buffer for exchange

Procedure

1. Remove column's bottom closure.
2. Loosen cap.
3. Place column in a 2.0 mL microcentrifuge collection tube.
4. Centrifuge at 1500 x g for 1 minute.
   • Removes storage solution.
5. Discard storage solution.
6. Mark orientation of column in centrifuge so that the column is placed in the same orientation each time.
7. Add 300 μL Buffer.
8. Centrifuge at 1500 x g for 1 minute.
9. Discard buffer.
10. Add 300 μL Buffer.
11. Centrifuge at 1500 x g for 1 minute.
12. Discard buffer.
13. Add 300 μL Buffer.
14. Centrifuge at 1500 x g for 1 minute.
15. Discard buffer.
16. Add 300 μL Buffer.
17. Centrifuge at 1500 x g for 1 minute.
18. Discard buffer.
19. Move column to new 2.0 mL collection tube.
20. Apply 30-130 μL of sample to top of compact resin bed.
    • For volumes less than 70 μL, apply 15 μL stacker of ultrapure water or buffer to top of compact resin bed after sample has absorbed to improve sample recovery.
21. Centrifuge at 1500 x g for 1 minute.
22. Discard desalting column.