CALCULATE VOLUME OF ASSAY REAGENTS IN 3 ML IF MOLARITY OF THEM ARE GOING?

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urmil
urmil's picture
CALCULATE VOLUME OF ASSAY REAGENTS IN 3 ML IF MOLARITY OF THEM ARE GOING?

 A 3 ML REACTION MIXTURE CONTAINS
20 mM ALPHA KETOGLUTARIC,
5mM  PHOSPHATE BUFFER 7.0,
10 mM NADPH (to be added at time to start d rxn)
AND .1ml enzyme.
how to calculate the volume of these in a 3 ml mixture?

hcheung
hcheung's picture
Your question is unclear. The

Your question is unclear. The volume of each component is 3 mL in the 3 mL solution.
Are you trying to make the solution? It looks like the concentrations of the components are the final concentrations. To calculate what volume of stock was originally added:
C1V1= C2V2
(20 mM)(3mL) = [insert the stock sol'n concentration] V2
V2 was the volume of stock used to make the solution.

Sushma Dagar
Sushma Dagar's picture
 Hi Urmil,

 Hi Urmil,
 from your question it seems that these are the final conc. of your compounds. As hcheung suggested, you can use simple C1V1 = C2V2 .
we generally call it as N1V1=N2V2 and all you should be having stock solution of all your chemicals.

Now N1= molarity of your stock
V1= you want to find out
N2= final conc. i.e. 20mM in case of ALPHA KETOGLUTARIC
V2= 3mL.  Calculate for all the chemicals.

urmil
urmil's picture
hi.hcheung and Science rocks

hi.hcheung and Science rocks
No, the volume of each component in reaction mixture is not 3 ml but its has to be find out.
3 ml is the total volume of assay mixture which contains these reagents whose molarity is known but i dont know how to calculate the indiviual volume in a 3 ml reaction assay. i.e
20 mM KETOGLUTARIC ACID   - X ML
10 mM naph                                  - Y ML
5 mM PHOSPHATE BUFFER    - Z ml
total assay mixture                   --------------
                                                         3 ml
 these x, y, z has to be known.
you can syggest me the alternative proceedures

Sushma Dagar
Sushma Dagar's picture
 Hi Urmil,

 Hi Urmil,
We are not saying that the volume of each component is 3mL. You make the stock solutions of all your chemicals separately.  For example, your make the 1 molar stock solution of naph.
now you can calculate how much volume you have to add in 3mL of solution. Now you use this formula of
N1V1=N2V2 .
So the important thing is that first you have to make the stock solutions of all the chemicals and then add the required vol and raise your solution volume to 3mL by using milliQ.