CaCl2 Competent Cell - (Oct/25/2007 )
I was used to make competent cell by cacl2 but for a log while i did not perform such practice. Can some body give me opinion how much volume of 100 mM CaCl2 used to suspend the pellet twice. and what percentage of glycerol should be used to store the cell at -70.
I shall be very thankful to you
-julebo-
I use 60mM cacl2 and 15% glycerol.
For 50ml culture 2 washes with 10ml of ice cold solution of the above then i resuspend in 2ml of the cacl2 solution.
Hope this will do.
-newarray-
while washing how much time did you keep the pellet suspended in CaCl2........30 minutes is it necessary?????
QUOTE (newarray @ Oct 25 2007, 01:09 PM)
I use 60mM cacl2 and 15% glycerol.
For 50ml culture 2 washes with 10ml of ice cold solution of the above then i resuspend in 2ml of the cacl2 solution.
Hope this will do.
For 50ml culture 2 washes with 10ml of ice cold solution of the above then i resuspend in 2ml of the cacl2 solution.
Hope this will do.
-julebo-
I keep 30 min on ice. In this way i always get high competency.
-newarray-