I have performed many minipreps in the past and have had no problems until now. I am currently using the E.Z.N.A Plasmid Mini kit to miniprep pET28 (b), after the miniprep I nanodrop my samples and see DNA ~50ng/ul.
After the miniprep I transform 1ul into 50ul of competent e. coli cells, let that sit for 10 minutes at room temperature. I then plate all of that onto LB miller Kanamycin plates, let them sit to dry and then incubate the plates at 37 degrees celcius overnight.
In the morning I have no colonies on the plate.
I have tried using multiple kits and nothing has changed. I have transformed other plasmids into these cells so I know the cells are working. I think that it is something wrong in my miniprep. Has anyone else had this problem? Any help would be greatly appreciated!
Hi Amanda,
Is that really how you are doing your transformation? It is a very sub optimal method if it is. You probably can have some success as you are adding a vast amount of DNA to the cells. I've attached a link to a standard transformation protocol, I suggest that you follow it. You should then get much better transformation efficiencies. If you still have problems after that let us know and we can investigate further.
-Richard
https://www.addgene.org/plasmid-protocols/bacterial-transformation/
I agree with Richard, your protocol seems not to be that efficient. By the way: use freshly prepared competent E. coli cells. This might help, too.
Cheers, Christian
Thank you so much, you were right. The transformation was not efficient, I followed the protocol above and everything turned out beautifully. Thank you again for the help!
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