Knight:Centrifuge desalting/Zeba columns
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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
- Remove column's bottom closure.
- Loosen cap.
- Place column in a 2.0 mL microcentrifuge collection tube.
- Centrifuge at 1500 x g for 1 minute.
- Removes storage solution.
- Discard storage solution.
- Mark orientation of column in centrifuge so that the column is placed in the same orientation each time.
- Add 300 μL Buffer.
- Centrifuge at 1500 x g for 1 minute.
- Discard buffer.
- Add 300 μL Buffer.
- Centrifuge at 1500 x g for 1 minute.
- Discard buffer.
- Add 300 μL Buffer.
- Centrifuge at 1500 x g for 1 minute.
- Discard buffer.
- Add 300 μL Buffer.
- Centrifuge at 1500 x g for 1 minute.
- Discard buffer.
- Move column to new 2.0 mL collection tube.
- 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.
- Centrifuge at 1500 x g for 1 minute.
- Discard desalting column.