Plasmid size problems!
Posted 17 June 2010 - 05:58 AM
Im having a problem stuffing a rather large (14kb) plasmid into my competant E.coli. The concentration of the plasmid is around about 500ng/ul and im using 2 to 5 ul per 50ul of cells. The cells are ultracompetent XL10 Gold cells. I dont use B-ME but maybe i should. Does anyone have any suggestions as to how to solve this problem?
Posted 18 June 2010 - 07:39 AM
Posted 18 June 2010 - 07:46 AM
Posted 25 June 2010 - 10:27 AM
will be obtained per microgram plasmid added). A quantity amounting to 1 or 2 ng plasmid is usually
sufficient. It is important, however, to minimize the volume of the introduced DNA, to avoid overly
diluting the competent cells. A 1:5 ratio of DNA to cells gives good results."
so, you may wanna double check the plasmid amount you add to the transformation reaction.