Primer dilution and calculation - (Jan/13/2007 )

I just need to make sure these values are correct before I carry them out. Can someone please confirm them for me? Thanks.

I am using some primers for a PCR experiment.
On the tube, the original stock of Primer X (from the company) = 28.5nmol = 0.19mg in a 500ul stock
This gives the original stock concentration as 0.057 nmol/ul

I have to do a 1:10 dilution to make an Intermediate stock. This gives an intermediate concentration of 0.0057 nmol/ul
I want 100ul of this Intermediate stock.
C1V1 = C2V2
(0.0057*100) / (0.057) = 10ul pulled from original stock diluted in 90ul H2O to make 100ul of my Intermediate stock.

From this Intermediate stock, I need to make a 5 pmol/ul User Stock, with final volume at 100ul.
0.0057nmol/ul = 5.7pmol/ul
C1 = 5.7 pmol/ul
V1 = x
C2 = 5pmol/ul
V2 = 100ul
(100)*(5) / 5.7 = 87.7ul of Intermediate Stock into 12.3ul H2O

Is this all correct? My user stock is what I'll be using for the expt. but I was told to do a 1:10 dilution in the middle. Is that routine when primers are used?

Your calculation is correct.

The routine I use needs no calculations.

When I get my primer which comes as lyophilized, I resuspend it to 100 uM/L concentration. For example, for an amount of 28.5 nM of lyophilized primer (the amount is provided by the vendor), I add 285 ul water or TE buffer and get 100 uM/L stock solution. To prepare 10 uM/L (or 10 pm/ul) working solution, I take 10 ul stock solution and add to 90 ul water. As you can see, no calculations are needed.

We do it exactly as pcrman has described. Its so simple.

Thanks for the confirm. I don't think any of the primers we are using are lyophilized because we always have to do a dilution, which is not too hard but has to be done.

Comparing your 28.5nM (0.0285 pmol/ul) example primer to my primer concentration of 57000nM (57 pmol/ul), the vendor stock is lowered to a 100 pm/ul stock solution. From this stock, you're preparing a working solution. In the same way, I am lowering my vendor stock to a 1:10 stock solution and preparing a 5pmol/ul working solution from that. Because the primer from the vendor is higher in my case, I will have to dilute it down. I guess this is the reasoning behind the 1:10 dilution.

Also, you are resuspending your vendor stock in 285 ul water or TE buffer. If this resuspended stock solution is used for PCR and you make up a 10pmol/ul concentration, how much final volume are you making up for each case? I was trying to calculate it out, but not sure how much final volume you are making up, so could not figure out how you got to your resuspension value of 285ul. In what I described, I am making up 100ul final volumes for each step. Also, how much total volume does your lyophilized primer come in? Ours are usually ordered by 500ul.

We get primers lyophilised so, we add the required amount of TE/water to make a stock sol. of 100pmol/ul. So the final volume of every primers stock volume varies.

I am using 10pmol for the PCR so I would dilute stock 10x. = 1+9 or 10+90.