mimiW

Hi guys:

I am new to mol gen and I have been encourtering trobles with chemical transformation lately. However, after a couples trys, i finally got it to work, but I am not sure the colonies are what i think they are. So i am thinking that i should check the for the presence of the plasmid, to see if the colonies are what i think they are.

I am a bit stuck on how i should check for the plasmid, my understanding is that I inoculate the colonies in LB with appropiate antibiotics, then preform a mini prep to isolat the DNA, then run a gel to check for the presence.

I am not sure if that is the way.

Thank you

I am having problems with my assays, my blanks, which only contains water instead of the enzyme i am using, reads often in thousands of counts( in CPM), even when i leave them overnight to avoid chemo-luminescence. I am not sure where i went wrong in the process because this is not consistent, sometimes i get around hundreds and sometimes below 100, which is the desired value.

What really troubles me is that i often get my activities around thousands as well, usually in between 5000 to 15000 depending on the concentration and the amount of the enzyme  i add. So if my blanks are at the value of thousands, say 2000, from my last assay. I am not even sure if i should trust my readings as real enzymatic activities.

I use H3_glutamate

please give me some insights and suggestions.

Cheers