I am setting up an RT-PCR experiment for the first time and I am trying to get the calculations for making up my working primers.
This is what I have so far.
Forward primer:
initial concentration: 19.71 nmol
final desired concentration: 775 nMol
Final volume required of diluted oligo: 80 (I'll have 18 wells and requires 4 uL which would be 72 uL primers. I upped it to 20x4= 80)
Calculate:
Volume of oligo stock: 3.145611365 uL
Volume of diluent: 76.85438864 uL
Reverse primer:
initial concentration: 23.72 nmol
final desired concentration: 775 nMol
Final volume required of diluted oligo: 80 (I'll have 18 wells and requires 4 uL which would be 72 uL primers. I upped it to 20x4= 80)
Calculate:
Volume of oligo stock: 2.613827993 uL
Volume of diluent: 77.38617201 uL
I would round the volume of oligo stock and diluent to make it feasible.
You are making this harder than it needs to be and your math is wrong.
First, your initial primer is being listed as an AMOUNT of molecules (nano moles), not a concentration. You have to add buffer/water/whatever is standard in your lab to go from an amount to a concentration.
Second, you need to make more than the minimal volume of primers to have a chance of accurate measuring. Make somewhere around 500 microliters of the working concentration of each primer and freeze it in aliquots.
Most folks make a stock solution of 100 microMolar concentration of primers and dilute that again into a working concentration, typically somewhere around 2-10 microMolar.
You need to take into account the final volume of your PCR reaction to know if you have added the correct volume of a known concentration of primers. 4 microliters of each primer is not a typical amount, it's WAY too much volume. Most qPCR reactions are around 20 microliters total.
Last, most folks create a "master mix/cocktail" of all common reagents (buffer, enzyme mix, primers, etc.) and aliquot those across the reactions. That way you only have to add in the template.
Talk to folks in your lab. And double-check your math before you start any experiments.
Good luck!
PS Unless this is a first look at your data, 18 reactions is nowhere near enough to include the needed biological replicates, technical replicates, housekeeping vs gene of interest, treatments/tissue types, and standard curve samples.
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