I am trying to recreate a protocol that has PCR tubes with a total volume of 50 µL. Now in the paper some oligonucleotides are added in different molarities;
Enrichment oligo 400 nM
Coupling oligo 80 nM
Barcoding oligo 330 fM
Indexing oligo 400 nM
I5 Adapter oligo 400 nM
Now the company selling the oligo’s is selling them in quantities such as; 25 nmole or 100 nmole.
How do I figure out which nmole I need to buy and how much of the volume I should transfer to the PCR tube?
If there is any necessary information missing please let me know.
From my experience with Macrogen oligos, selected amounts affect only total volume of a stock solution. For example, 50 nmol of oligo gives 200-250 µL of 100 µM stock solution (exact volume varies in some range as there is an uncertainty of the product yield after a synthesis and purification, but is usually given by the manufacturer). 100 µM is quite a high concentration, so for the working solution it is usually ten times diluted (10 µM).
With a 10 µM working solution, from your example (enrichment):
1) 10 µM/400 nM = 10 µM / 0.4 µM = 25
2) 50 µl / 25 = 2 µL
So, to have 400 nM concentration in 50 µL you need to add 2 µL of the working solution. Same way you calculate other volumes and then adjust the final volume up to 50 µL.
c1v1=c2v2
You know your desired final volume & final concentration. Most folks dilute their primers to an initial working concentration of 10 micro molar; so that gives you an initial concentration. Solve for v1.
If the volume you get for v1 is too low (less than 2 micro liters), then start with a more dilute working concentration (say 1 micro molar).
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