Plate Assay active ingredient dilution - Dilution problem (Nov/30/2010 )

Hi People

I am running a 96 well plate growth curve assay testing the inhibitory effects of a substance against a number of bacteria, and i cant seem to figure out the correct stock/working concentrations of the substance.

For each column i want to have different concentrations of my substance, 1ng, 10ng, 25ng, 50ng , 75ng and 100ng.

The problem arises when i need to add the same volume of substance to each well (as opposed to just increasing concentrations of the same stock solution, the reason for this is it is soluable in acid and will influence the growth so i must normalise with a control).

So for instance if i set an arbitrary volume of 10 microL of my substance for each well (with 10microL of the acid without substance as a control)

The total volume i will be using is 250 microL in each well, im aware that my stock solutions will need to be different for each of the desired concentrations but am unsure of how to calulate this.

I have the substance powdered and purified so i can make up any reasonable volume for stocks.

Any help with workable volumes (more than microL) would be greatly appreciated, the only set figures are total volume of 250 microL and 1-200ng concentrations in each well at a fixed vol for each.

Sorry if this question is convoluted, i have attached a figure which may help to show what i need.


Chris
make up

So if I understand this right, you already have 250ul in each well and want to add 10ul of a drug stock to each well at varying concentrations. since all you gave was amounts (1ng, 10ng, 25ng, 50ng, and 100ng) I'm going to assume you meant that you want each well to have a final concentration of 1ng/ul, 10ng/ul, 25ng/ul etc.

so if you add 10ul to your 250ul in the well you will end up with a total of 260ul. that means you need enough drug in 10ul to account for all 260ul of volume. 260ul/10ul=26 you need to make your stock for each well 26x in concentration (so 26ng/ul, 260ng/ul, 650ng/ul, 1300ng/ul, 1950ng/ul, and 2600ng/ul)

so in the end (i'll use the lowest dose for an example) you will add 26ng/ul*10ul=260ng drug to each well. 260ng/260ul gives a final concentration of 1ng/ul.

Hope this wasn't too confusing of an explanation.