Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

how to make a working solution of a primers for pcr reaction?? - (Feb/06/2013 )

I have a big problem because I don't know how I need to do a primer dilution....
The lyophilized concentration of primer is 20 pmol and the concentratios is 7 ug and OD is 0.2.

so i have no idea how to make a stock solution and working solution for my pcr reactions

Some body pls help me...
Thanks
Attached File

-gourav88-

How do you know the conc. of lyophilized primer is 20pmol? Is it mentioned in the document that you attached? To make a stock of desired conc. you need to dissolve the lyophilized primer in autoclaved water. And from that stock you take the required amount to make up the final conc. of primer in PCR reaction. check the link for calculator-

http://www.exiqon.com/ls/Pages/ExiqonDilutionCalculator.aspx

-neuron-

The data sheet you attached says: "These Primers and Probe are synthesised and purified by PAGE. They are supplied as 0.2 OD (6.6 μgs) lyophilized. To reconstitute, dissolve in 20 μl sterile water, check concentraion by OD before use."

So, according to this you just have to add 20µl of water. The predicted concentration will be 330ng/µl (6.6µg/20µl), but you should confirm by nanodrop or similar methods. From this dilute to your working concentration.

I'm not sure where you get that "The lyophilized concentration of primer is 20 pmol" On that, a very important point. 20pmol is NOT a concentration, is total amount. Same goes for 7µg (or actually 6.6µg according to the attached document), this is NOT a concentration but an absolute amount. You can make this up to the required concentrations by adding water (or TE).

Back to what the data sheet suggests, of dissolving in 20µl of water, and assuming that 6.6µg of primers are indeed 20pmol. Primers dissolved this way will be at a concentration of 1µM (20pmol/20µl).

Hope this helps.

-almost a doctor-

That is a really strange stock primer concentration. What company are you getting your primers from? Did you design them or are they predesigned? I tried searching for "GeneI" (the name at the top of your .pdf), but nothing came up.

-jerryshelly1-

it's this apparently (GeNei Bangalore)...

http://www.merckindiawebapps.com/merckindiawebsites/merckmillipore/index.html

-Tabaluga-