Centrifuge desalting/Zeba columns-PDF

Overview

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

Materials

• Zeba Desalt Spin Columns, 0.5 ml from Pierce
• Variable-speed bench-top microcentrifuge
• 2.0 mL collection tubes
• Buffer for exchange

Procedure

1. Remove the column’s bottom closure.
2. Loosen cap.
3. Place the 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 the orientation of the column in the 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 the column to the new 2.0 mL collection tube.
20. Apply 30-130 μL of sample to the top of the compact resin bed.
    • For volumes less than 70 μL, apply a 15 μL stacker of ultrapure water or buffer to the top of the compact resin bed after the sample has been absorbed to improve sample recovery.
21. Centrifuge at 1500 x g for 1 minute.
22. Discard the desalting column.