How should a stock solution and work solution with tree pollens be prepared?
Dear honored scientists,
For an herbal extraction that contains multiple compositions (like different kinds of antioxidants and lipids), there is no information about molecular weight. How can I calculate the concentration of the stock solution and prepare different working solutions?
For pure drugs, I use the GraphPad molarity calculator web. First, I calculate the stock solution concentration and, after that, the "dilute a stock solution" part calculates my doses.
Hi (again) Elham,
I would describe how you obtained the stock solution (amount of raw material before extraction + volume of solvent) and note for the work solutions what the dilution factor is. So for example 'Extract of 5 grams of willow bark in 10ml of distilled water' (+extraction protocol), and then for the working solutions note "1:10" willow bark extract, "1:50" willow bark extract, ... etc.
You could also perform the extraction, and characterize some aspects of the obtained stock solution, for example protein content (Bradford or BCA assay).
I hope these suggestions are helpful,
Good luck,
Jasper
Jasper B.J. Kamphuis Hi many thanks for your helpful answers
I am working with a kind of fruit tree pollen and for my first test, I solved 0.1 g in 10ml PBS and I didn't warm it, I only put it in the fridge (I only followed my labmate protocol). After filtering the solution for preparing my doses (10, 20,500,.....) I pick 1 λ from my stock and solve it to 999λ complete medium for 10 and did this for other working solutions too. It was difficult, but however I did that.
Jasper B.J. Kamphuis I'm not sure what I did was correct or not because I cant measure stock concentration!!!
I appreciate your guidance.
Dear Elham,
I'd just say your concentration is 10mg/ml (from 100mg/10ml) (noting that insoluble parts are lost, you have a solution obtained by 100mg pollen in 10ml PBS). Then, if you want to make dilutions, you dilute it 10 times (1 part stock, 9 parts PBS) for 1mg/ml, and continue dilutions like that, either from the stock, or from other dilutions (for example to go to 0,1mg/ml, you can dilute the first dilution (1mg/ml) 10 times, instead of diluting the stock a 100 times, this is often easier and more precise, because you evade pipetting tiny volumes). I didn't understand what you mean when you diluted the dilution a 1000 times in complete medium to obtain '10', the protocol asked for 10ng?
Hope this helps.
Kind regards,
Jasper
Hello dear Jasper B.J. Kamphuis ,
So I had a mistake. I want to make 10 μg/ml, 20 μg/ml, 100 μg/ml to find IC50. For cell treatment, I usually pick my desired volume from stock and dilute it in the medium (making a total volume of 1 ml), then I pick 200 μl and treat cells in the 96 well plates.
Could you please tell me how to make 10 μg/ml from 10mg/ml with a volume of 1 ml?
If I dilute my stock with PBS ( for example, 1mg/ml )should I add it directly to the well and after that adding medium?
Sorry, I asked many questions. Thank you for your help.
Well, your starting dilution is 10mg/ml (from 0,1g in 10ml), so a 100 times dilution (1 part stock + 99 parts medium) would give you 100μg/ml, you could for example prepare 2ml of this (depending how many dilutions you need to make), that would be 20μl starting dilution + 1980μl medium, we'll call this 'dilution 1'. Now to get to 20μg/ml you can make a further dilution of 5 times from dilution 1, for example 200μl dilution 1 + 800μl medium (say dilution 2). For 10 μg/ml you'd have 100μl dilution 1, 900μl medium (say dilution 3). Now you have a total of 1700μl (2000μl-200μl-100μl) dilution 1(100μg/ml), 1000μl dilution 2 (20μg/ml), and 1000μl dilution 3 (10μg/ml). Now that you have your dilutions, and they're already in the appropriate medium, you can use them to treat your cells.
Good luck!
Kind regards,
Jasper
P.S. edited to correct an error
Dear Jasper B.J. Kamphuis
Thanks for your help, I completely understand what should I do. I will try my best.
Kind thanks,
Stay safe and well.
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