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### Topic: Designing an assay experiment - dilution problem  (Read 176 times)

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#### yogurtspoon

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##### Designing an assay experiment - dilution problem
« on: September 11, 2020, 10:32:12 PM »
So, I was given this problem a little over a week ago. My TA helped me start it, I continued by myself & almost finished it, now I'm looking over it and I'm having trouble making sense of what I did. I also think I might have mixed up some numbers, but I'm not sure.

Here is a condensed version of the problem. (It's for my biochemistry lab but I don't think the skills it's testing are specific to biochem? Feel free to lmk if I'm posting on the wrong page though.)

"You are given a 1840 μM stock solution of inhibitory-compound X (I will call this stock A). You plan to perform a drug titration assay using X. You decide to use the following compound X concentrations in your assay: 100 nM, 250 nM, 500 nM, 1 μM, 2μM and 5 μM. The total reaction volume for each translation assay is 25 μL. Your inhibitor content for each reaction tube must be 5 μL. All other dilutions after making your initial stock of compound X in DMSO MUST be in water to avoid total protein unfolding. Make a table of how you would design this experiment and show all your work."

I think the plan for my "experiment" was to make 25 mL of 5 μM X to use as a new stock solution (I will call this stock B). I'd use the first 5 μL of stock B for the 5 μM test tube. Then I'd keep diluting the stock to the other concentrations, separating 5 μL into a tube each time.

Here's what I have written down in my notebook (asterisks for side-notes, underlined all my confusions/questions):

For the 5 μM tube
C1V1 = C2V2
(C1)(5 μL) = (5 μM)(25 μL)
C1 = 25 μM *I have this number labeled "concentration of X stock needed," not sure what I'm talking about though*

C1V1 = C2V2
(1840)(V1) = (25 μM)(20 μL)
V1 = 0.27 μL *labeled "to make 25 μM working soln" Assuming that means stock B?*

*At this point my TA said that 0.27 μL is too small to pipet (for our lab's equipment at least, idk). So I decided to do a 10-fold dilution of the original 1840 μM stock solution, so that there'd be more volume.*

C1V1 = C2V2
(184 μM)(V1) = (25 μM)(20 μL)
V1 = 2.7 μL compound X
*Under that, I wrote "plus 17.3 μL water" and I'm very confused by that. That would make a 20 μL stock solution. Not even the 25 μL I said was enough for all of the reaction tubes. I thought it was a mistake, but I'm also pretty sure my TA gave me that 17.3 number so Idk for sure. If the calculation is supposed to say 25 μL, then do I have to fix this one and all the following calculations?*
*Also, this is the only work I have written down for the 5 μM tube. I'm wondering whether I'm supposed to calculate for the 5 μM tube the same way I calculate for the following tube...*

For the 2 μM tube
C1V1 = C2V2
(C1)(5 μL) = (2 μM)(25 μL)
C1 = 10 μM
*Tbh I don't really get remember point of doing these calculations? It seems kind of backwards to me, if that makes sense. The fact that we're solving for concentration instead of volume first. What does C1 represent in this step? Is that the concentration I need to dilute stock B to?*

C1V1 = C2V2
(25 μM)(V1) = (10 μM)(5 μL)
V1 = 2 μL X stock soln + 3 μL water
*With an arrow pointing to the second CV=CV equation, I wrote "use previous stock". So, is 25 μM the concentration of stock B? Also, should I do this calculation for tube 1 as well? If I do, then I get 5 μL stock + 0 μL water. Is that reasonable?*

I did analogous calculations for the remaining tubes, this is was what I got:
1 μM: 1 μL + 4 μL water
500 nM: 0.5 μL + 4.5 μL water
250 nM: 0.25 μL + 4.75 μL water
100 nM: 0.1 μL + 4.9 μL water
*No notes except the last three seem like suspisciously small numbers to me. Is this experiment still reasonable?*

This was super long-winded, thanks to anyone who gives it a go.

#### AWK

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##### Re: Designing an assay experiment - dilution problem
« Reply #1 on: September 12, 2020, 04:10:25 AM »
If I understand correctly, you need the above-mentioned concentrations in the total reaction volume 25 μl so your highest concentration after dilution is 25 μM. With a sret of micropipettes you can dou your dilution within 1 ml volume of diluted solution.
As for DMSO - it cannot be easily removed from the solution, but DMSO will also be diluted ~ 70-3600 times.
When planning, you need to plan the experiment optimally for your set of micropipettes, taking into account their increments.
« Last Edit: September 12, 2020, 04:26:59 AM by AWK »
AWK