transformation and protein purification calculation help; very basic - (Nov/26/2013 )
I have two questions, both a bit lame.
1. I want to do my first transformation. I dissolved my DNA off a paper disc following instructions from the company who made it. My nanodrop reading (I put 1ul of my DNA on it) was 20.4 ng/ul. I reckon that I got about 90ul in volume so 1.8ug? I now want to transform this DNA into E.coli using KCM. It was recommended to me to transform 100ng. Would I be right to use 5 ul DNA? If I have this wrong please may I have an example how to go about it?
2. My next question is that I have a protocol to do with purification and I am to lyse the cells first. I am to add about 15mL suspension buffer......what does it mean to add 1-X Triton (1 uL.mL-1 )? I am confused about how much of Triton to add. How do you interperate (1 uL.mL-1 )?
I don't know what KCM refers to. The amount of DNA used for transformation is unimportant, so long as there is sufficient to transform at least one cell. For most chemically competent cells, you should use less than 5% additional volume of DNA solution when transforming. Usually 2-3 ul is sufficient with good competent cells.
(1 uL.mL-1 ) means you should add 1 ul of Triton for each milliliter of volume of your suspension buffer. If you are using 15 ml, then you should add 15 ul of triton.
Thank you so much veteran, that was very helpful :-)
Just one more thing....why bother putting -1
Would that not mean 1mL/10? ie 100?
Sorry, I am confused about that bit, I am not used to prefixes at all and get the heebies every time I see them.
You should read that as "per milliliter". This is an exponent on the unit. Just as 2-1 is 1/2, the unit ml-1 is 1/ml. So, 1ul.mL-1 is the same as 1 ul/ml or 1 microliter per milliliter.
Ah ok, thanks yet again