I am working on the DNA Barcoding of the olive in Pakistan. I have just ordered the primers. Please guide me how to make the solution of the primers and how to make the working dilutions out of it.
Dear Rahil, you can dissolve your primers as described in synthesis sheet. The described amount of 1xTE or sterile ultra pure water can added to make 100 uM concentration of primer. Then you can dilute from this stock solution to 10 uM for PCR rxn or other purposes. Store your primers -20 C
Another way is dissolve your primers in 1xTE or sterile ultra pure water in 200-300 uL than measure absorbance of primers at 260 nm. After that calculate the concentration: OD260nm x 33 x dilution factor. This gives you ng/uL concentration primers than you can dilute from this concentration for further experiments.
Best regards.
http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html
When I get my primer which comes as lyophilized, I re-suspend it to 100 uM conc. 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 stock solution. To prepare 10 uM working solution, I take 10 ul stock solution (at 100uM) and add to 90 ul water.
Hi Irina,
I think you meant 28.5 nmoles of lyophilized primer from the vendor (instead of 28.5 "nM"; which is a concentration). Just a detail - but one which does get confused often...
Also - if wanting more detail and want to use extinction coefficients etc. for this - see the attached tool which I made for this purpose as well...
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