The data sheet provided by company has mentioned that dissolve the primer in 200ul of sterile water or TE and it gives 242.65pm.
How to prepare the working stock? Which concentration of the primer will be best to use it?
In agreement with Ali above,
If you added 200 uL to the dry primer lyophilate and obtained a concentration of 242.65 pmoles/uL (or 242.65 uM), that means the company originally sent you 48530 pmoles (e.g. 48.53 nmoles).
If you would have added 485.3 uL to the original dry lyophilate, you could have obtained a more usual stock concentration of 100 pmoles/uL (100 uM) directly instead.
Then, from there, as Dr. Ali Mahmoudpour has stated above, you can make 10 uM primer pre-qPCR stock by a simple 1:10 dilution of the 100 uM stock. This 10 uM stock then is a good concentration to pipet from in order to accomplish whatever concentration you desire in your qPCR reactions. Proper calculation of primer concentrations in solution based on MW and extinction coefficients can be accurately accomplished using the attached Excel file tool I created to use for this. The confusing elements of this seemingly basic maneuver are many - but the attached file hopefully takes away all the mystery.
The main reason to use a file like this is because the company often sends +/- a certain amount from the amount stated. One tests this by first making a 100 uM stock of primer solution, then tests a 1:100 dilution of that (1 uL + 99 uL TE pH 8.0; achieving a theoretic "1 uM" concentration) on a spec to see what the real concentration is. The spec is blanked with TE pH 8.0 before taking the reading of course. If you find, for instance, that instead of a 100 uM solution, you actually have an 87 uM solution, then this file tells you what volume of TE to add to the remaining 484.3 uL (in this case) to exactly achieve a 10 uM pre-qPCR primer stock. If one assumes one is starting with a 100 uM stock without checking that on a spec first, then one will be off in one's calculations as to what volume of TE should be added to obtain the 10 uM pre-qPCR primer stocks...
The attached file attempts to take all of the "trickiness" out of this process - but can only be used if the company provides you the MW and extinction coefficient of the oligo they sent you. In the file attached, I have entered values in cells I2 and I3 as stand-in values for your situation - so you will have to input the real values there with the values you received from the company. Also be certain to use the correct units for the extinction coefficient. E.g., in the attached file, the extinction coefficient is assumed to be expressed in units of M-1cm-1 in order for the file to work correctly. I.e., if the company gives you a mM-1cm-1 coefficient of 220.2, you would simply convert this value instead to 220200 in order for the file to work correctly. As well, cell I4 is in terms of nanomoles (nmoles) instead of picomoles (pmoles), so you would use the value "48.53" in your present case.
Dear Raksha, this can be solved by one another simplified way, as preferred and favored by students and new learners and is also quite easy to understand.
You have in original 242.65 pm / uL with 200 uL diluent, which in other words is 242.65 uM, as explained above by respected colleagues. From this to make a stock of 100 uM we will use the universal equation of V1C1 = V2C2, which I hope you are already aware of and it very simply makes the entire job understandable. In this case we have V1, C1 and C2 already in our hands, all we need the second volume or V2 require to make 100 uM so;
V2 = V1 x C1 / C2 = 200 x 242.65 / 100 = 485.3 uL, so add this into your original stock will give 100 uM C2. Finally, to recheck this just assume that you do not know the C2, (which is 100 uM in our case). So now, C2 = V1C1 / V2 = 200 x 242.65 / 485.3 = 100 uM, which means your calculations are perfect and no worries. Thus, you can see that just by playing with this equation it is very easy to get what you want in terms of volumes or the concentrations.
And whatever concentration of working primers you need from this 100 uM stock can also be calculated by the same equation. Or simply take 1 uL of it into 99 uL water or TE to make 100 uL working aliquot, which is 1:100 dilution or 1 uM (1 pm / uL) concentration. I hope you got it....
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