How to quantify virus by PCR - (Aug/29/2012 )
You got 40 ng/ul RNA. You needed 3 ng, that would mean to pipett 0.075 ul. That's not possible, right. How exactly did you diluted?
You wrote "i did a diltution of 40 ng/µl to 0.15 µl to obtain the 2 µl volum used", that's kind of confusing, what concentration of RNA did you diluted to? (not the final concentration in the PCR reaction, that is absolutely unimportant value right now)
If you diluted RNA to 0.15ng/ul before you pipett to PCR, that would be like 266 times, and that's too many. You say that you used 2 ul to PCR, that would mean you should have your RNA concentration 1.5 ng/ul, that means diluting 1 ul of RNA <40ng/ul> in 25.7 ul of water. Any other dilution would be wrong.
As for lyophilised primers, you just add water or buffer you use. Common storage concentration is 100uM (100 pmol/ul). There should be come information from your primer provider, nmol, ug or OD values of the synthetised primer. Mostly there is already an information how much water/buffer to add to get certain concentrations.
My dear i diluted 40ng/µl to 1.5 ng/µl then to 0.15ng/µl
that is right they say i should dilute it in 550 µl TE buffer with PH 8. The question if i dilute it in RNAse free water . will it affects the PH of storage as the water is not similar to TE buffer in a term of PH .
Primers are usually diluted in TE or 10mM Tris pH 8. Higher pH is better because it prevents degradation. Unbuffered water can vary in pH in time. Technically your buffers should be made with nuclease free water, so it would be sterile and have the righ pH.