Matt Gethers/CRI, Thailand/Labwork/Dialysis
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- Get dialysis tubing and cut more than enough to hold the volume you intend to dialyze (2 ml/cm of tubing). Be sure the tubing has a pore size appropriate for the molecular weight of your protein.
- Fill a beaker with RO water and wash the tubing. Be sure to separate the sides of the tube so the inside can be washed.
- Use a clamp to seal one end of the tubing. Be sure the tubing is flat and is in the middle of the clamp.
- Fill the tubing with your sample and clamp the open end.
- Fill a beaker with 1 L of dialysis buffer (see contents below) and place the clamped tubing in the beaker.
- Add 1 ml 1 M DTT to the liter of buffer.
- Place the beaker at 4oC on a stir plate over night.
- When dialysis is complete, unclamp one end, pour the sample into a tube and place on ice.
- To estimate the final concentration of the sample, divide the moles of salt both in the sample and the buffer by the total volume (1 L).
|5 M NaCl||20 ml||50 mM|
|500 mM EDTA||4 ml||1 mM|
|1 M DTT (Don't add to master)||1 mM|
|1 M Tris pH 7||100 ml||50 mM|
|100% Glycerol||100 ml||5%|
|H20||Up to 2 Liters|
Be sure to add 1 ml 1 M DTT to 1 Liter when ready for use.
I made 2 liters of buffer as per the protocol above. I'm not prepared to dialyze samples yet, though, so I've placed the 2 liters in the four degree. No DTT added.