Chemically competent cells? - (Jun/14/2007 )
How important is it to incubate the cells you want to make chemically competent 20 minutes on ice? Can they be incubated longer? It's a step in between resuspending in magnesium chloride and calcium chloride. I happened to have left them incubating on ice longer than 20 mins so I am wondering is I made a mistake there.I will do a control transformation, yes.
-smoochiepie79-
QUOTE (smoochiepie79 @ Jun 14 2007, 01:05 PM)
How important is it to incubate the cells you want to make chemically competent 20 minutes on ice? Can they be incubated longer? It's a step in between resuspending in magnesium chloride and calcium chloride. I happened to have left them incubating on ice longer than 20 mins so I am wondering is I made a mistake there.I will do a control transformation, yes.
Meh. Making competent cells (in my experiences) is relatively flexible. Grow some cells to about log phase (OD=0.4-0.6), wash them several times, and keep them cold on ice whenever possible. I usually put them in the fridge overnight before aliquotting and freezing. I never even use magnesium chloride. Again, science is like cooking. Just try to wash cells to get rid of the media, and put them in some glycerol so they don't lyse when you freeze them. That's it!
-Cheamps-
QUOTE (Cheamps @ Jun 14 2007, 07:44 PM)
QUOTE (smoochiepie79 @ Jun 14 2007, 01:05 PM)
How important is it to incubate the cells you want to make chemically competent 20 minutes on ice? Can they be incubated longer? It's a step in between resuspending in magnesium chloride and calcium chloride. I happened to have left them incubating on ice longer than 20 mins so I am wondering is I made a mistake there.I will do a control transformation, yes.
Meh. Making competent cells (in my experiences) is relatively flexible. Grow some cells to about log phase (OD=0.4-0.6), wash them several times, and keep them cold on ice whenever possible. I usually put them in the fridge overnight before aliquotting and freezing. I never even use magnesium chloride. Again, science is like cooking. Just try to wash cells to get rid of the media, and put them in some glycerol so they don't lyse when you freeze them. That's it!
Thanks! Why does glycerol help cells from lysing?
-smoochiepie79-
in this situation, the glycerol is intefering with the formation of large ice crystals. Ice crystals will form within a cell when the cell is frozen. If the crystals gets too big, it physically tears up the cell membrane, and the cell dies when defrosted.
-perneseblue-