I would like to ask that I have tried to make competent cells by 0.1MgCl2 and 0.1mM of CaCl2 treatments. My question is, what if I perform a real quick washing of bacterial pallet with 0.1mM CaCl2 before suspending to 0.1mM MgCl2?
Sequence of steps
1-Resuspension of the bacterial pallet into 0.1mM CaCl2, centrifugation at 4 degrees
2-Resuspension in 0.1mM MgCl2 and 15 mins of incubation on ice, centrifugation at 4 degrees
3-Resuspension in 0.1mM CaCl2 and 30 mins of incubation on ice, centrifugation at 4 degrees
4-Resuspension in 1ml of 0.1mM CaCl2 to make glycerol stock.
What do you think competent cells would take up DNA (plasmid) or not?
I think it works, I only washed CaCl2 for 2 times before, it works well.
Some lab using RbCl2 instead, I have tried it, it seems better.
You should check the concentrations of CaCl2 and MgCl2 needed.
0.1mM looks very low - is it supposed to be 0.1M?
Vincent Mulholland I am sorry, concentrations were mistakenly written as 0.1mM. Correct concentrations for MgCl2 and CaCl2 are 0.1M.
Hi Muhammad,
Have you tried washing the pellet with 0.1M CaCl2 before re-suspending them? Actually I always wash the bacterial pellet with 0.1M CaCl2 one time then followed by re-suspending them in 100uL of 0.1M CaCl2. It works better and the cell is competent enough.
Thank you.
Sarannia Thanganathan I have asked before MgCl2 treatment I have washed the cells with 0.1M CaCl2.
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