EL250 relectroporation - (Sep/17/2007 )
i am now trying to transform EL250 with circular herpesvirus. but i have no result.
the published conditions of electroporation are 1.75 kv, 25 uF and 200 oohm.
unfortunately, our electroporator can not provide all these conditions so i used 1.8 or 2.4 kv, 10 uF and 600 oohm's.
i prepare the competent cell myself.
now, i do not know if my EL250 strain is not good.
or, my preparations are not good.
or electroporation conditions are not suitable.
do you know a source for new EL250 strain.
I would first test the cells by transforming a plasmid you know is good to test whether the problem is with your construct or the cells/electroporator. Transform 0.1ng of the plasmid into 50ul of your competent cells and count the number of colonies you get back. If you have no colonies there is a problem with the cells or the electroporator, if you get 100 colonies or more then you have a competency of greater than 1x10^6 cfu/ug, which is pretty good and would suggest the problem is with your construct.
actually i did. and i have many colonies over 100. but i think there is no comparison between the 2 plasmids.
the control one is about 3kb but the used one is around 150kb (this is circular viral DNA).
another thing, after electroporation of the big plasmids, i have some yellow colonies on the plate (i use chloramphenicol) even it did not appear with the control plasmid.
How many nanograms of the 3kb vector did you transform, and how many colonies did you get back? This is relevant because it tells you how good your cells are at being transformed.
around 300 ng of 3kb vector and uncountable number of colonies.