Electroporation technicalities - (Jul/06/2007 )

Hi all cDNA gurus

I have a question about electroporation. When I set all the parameters (capacitance, resistance and voltage) I press those two red buttons and:

a) quickly depress them,

wait untill I hear buzzing and then depress,

c) wait for arcing,

d) depress them when I see smoke coming out from Bio-Rad machine

Please this is a serious question!!!


Rob

I'm sorry, what is your question again?

I wait for a short time (few seconds) while the machine buzzes before letting go of the buttons.

arching is bad. The cells are mostly dead after having been electrocuted. You have either too much DNA or your DNA is contaminated with salt. Wash your DNA with 100ul 70 % EtOH, spining down the mix after addition of the 70% ethanol to avoid the DNA from detaching from the tube wall.

Smoke from the machine is very very bad. Go inform your supervisor and get the machine to the shop.

We wait for 1-2sec after having the buttons pressed and release it after the buzz.

As perneseblue suggested if there is too much salt, it will spark. You need to precipitate the DNA before ligation or dialyse the salt from the ligation mixture. You could add a lower volume of ligation mixture or use more cells.

But if there is smoke from the machine, check with PI or call techincal support.