electroporation using Electroporator2510 - Time Const has just 2 msec (Mar/02/2007 )
I m using your Electroporator2510 to make electroporation using E. co//JM109 .pir .
The competent cell was made as usual: harvesting the OD600 0.6-0.8 cells ,
ice chilled 15 min, -1 C centrifuge for 5 min , then ice cold dd H2O washing 3 x ,
the final ca.100 microliter rest kept for electroporation. ( 1mm gap cuvettes, 1350 v ,
Pulse/Pushed 2x , “chg” appeared.)
My question is Why the actual Time Const has just 2 msec ( rather than usual
5 msec) ? When I used other competent cell such as EPI 300 ( Epicentre inc.)
The Time const is 5 msec. What causes this difference?
if DNA too less ? This " 2msec" gave no colony at all in plates
How to optimize the conditions to improve the transformation using this instrument?
It s a basic question , but important now.
Any suggestion is greatly appreciated.\\Have a nice weekend!
Sorry for the late response, In my experience working with HD5 alpha electrocompetent cells, generally I get lower transformation times for ligations, than controls (7msec for controls and 4 for ligations), so I think If you have high Ion concentration you get lower transformation times. But when I get lower times, my cells allways arce. So I do not get any colonies in my plates.
I do electrocompetent cells, harvesting the OD to 0.4 - 0.5, ice chilled 20 min, centrifuge 4ºC 5 min at 6000rpm , then washing with Ice cold water 2x, then washing 1x with glicerol 10%, after discard the supernatant from this step I resuspend in 2ml of glicerol 10%, and aliquote 200 microliter on 1.5 ml eppendorf tubes.
May be you should try the glicerol step to get longer times for transformation.
Hope that helps.