DNA amount for protein expression - protein His-tag (Jan/21/2006 )
I would like to purify a protein His-tag but normally (I just purified a protein once) to express the protein in competent cells I use 10 microliter of 50 microliter of my PCR product for the transformation of 100 microliter of cells.
This time I want to use the purified plasmid (containing the gene for the protein) for the transformation in taking 25 ng of it for 100 microliter of competent cells.
Do you think it is a right amount?
Thanks in advance.
I am not sure if I understand your application, I would assume that you would plate and screen colonies then choose a single clone to grow up and express the protein?? If my assumption is correct, then the way to determine the amount of DNA is to look at the colony forming unit for your competent cells or CFU. This is a number representing the #of colonies made per amount of DNA (usually ug) transformed you can use this to get a general estimate... Usually you don't have to be so quantitative though... just add DNA and plate dilutions of your transformation so that one of your plates has a reasonable number of colonies on it...
-ratio of DNA mixture to competent cells, usually no more than 10ul DNA for 100ul competent cells
-having too much DNA can be toxic and will reduce the transformation efficiency, in this case it may not matter, you are transforming a purified plasmid and almost all the colonies you get should be correct, reducing the efficiency should not matter...
-if you have limited DNA then I think you can get away with less than 25ng in this case... but I don't think it will hurt to use that much either... decide based on how much you have....