preparation of electrocompetant cells. - what about the density? (Aug/11/2006 )
i did the preparation all the washing and everything. but just eliquoted the cells and put at -80C not knowing how to dilute them...
i measured OD of 1/100 it was around 1. so i left cells undiluted. molecular biology says OD1 is too much i should dilute.
what shall I do now? i can't thow to dilute? will they be ok for electroporation? or too dense??
Usually we check OD before starting the washing steps. And for electrocompentent cells, I have made them even after the OD was between 1 - 1.1 . We dont check OD after the washing steps.
And when we finally aliquot them, we always use the same volume (if OD is higher or lower). So I would suggest u go ahead and try a control electroporation with supercoiled plasmid (eg. pUC or pBS) and check competency of cells before proceeding with ur actual electroporatrion.
hey, Kathy, if you are using the method we discussed via PM, you are NOT supposed to freeze those cells! those are to be made fresh immediately before electroporation and not to be stored...this could by why your final density is low and why they are popping, many of the cells will be dead because there is no buffer to sustain them through the temperature changes