Second Guessing myself on primer concentrations

[nebulus]

Hi all,

I have been preparing my primer solutions this way and need some help

Here is an example of 1 primer tube and how I've made a working solution

Primer M.W= 6,298.1
7.2OD=39.2nm=0.25mg=250ug

I first suspend the primers in 100ul of PCR grade H2O= 250ug/100ul=2.5ug/ul

The PCR optimization guide told me to add 1ul of (0.25ug/ul) working solution to my reaction.

To get my 0.25ug/uL primer working solution of 200ul total volume I take 20ul of stock and 180ul of PCR grade H20 v1(2.5)=200(0.25)

1.) I want to know what the uM concentration of this solution is, because the gene sequencing lab is requesing primer solutions of 2uM.

2.) Also my PCR optimization guide says primers should be between 0.1uM and 0.5uM final concentration in my PCR solutions. In my case 50uL total PCR reaction solution. What would be the final uM concentration for this primer?


Many many thanks

[danfive]

1. stock is 392uM; working solution is 39.2uM
If you or seq. lab wants working solution of 2uM change DF of stock.
392uM/2 = 196---> DF of 196 would give you 2uM (1ul stock into 196 total volume).

2. If you use 1ul primer working solution for 10ul reaction (DF is 10) so PCR concn = 3.92uM
If you use 1ul primer working solution for 25ul reaction (DF is 25) so PCR concn = 1.568uM