I have standard curve and know the plasmid size. How can I get the copy number? Can anyone explain details or give reference? It will be really great help.
You want the copy number of the plasmid? If you do an absolute quantification of a sequence on this plasmid, then you have the number of plasmids... so I don't understand the problem.
If you have quantified the DNA-concentration (w/v) of a plasmid prep and want to get the numbers of molecules, you just need to know the mass of a basepair (=660 g/mol, and 1 mol = 6.022E23 molecules). The rest is very very simple math. If you need some further help, here are two references showing and explaining the calculations:
qpcrupdate.net/changsoo-lee-ecoli-cnv-2007.pdf
http://nar.oxfordjournals.org/content/31/10/e56.long
I hope it helps.
Hi SM,
You said you were very new to PCR, I hope I can give you a proper breakdown of the calculations and how to obtain them. If I've understood your question right, did you mean to say that you have plasmid material that you want to use for your DNA standard curves and you want know how to calculate the copies/ul of your stock?
If so, this equation may help:
* copies/ul = conc. / mw
It is important that the stock is pure dsDNA (check A260/280 and A230/280 ratios) and generates the typical peak at 260nm (see image at http://biophy.uchicago.edu/spec.php).
To calculate concentration manually, first measure the A260 value (not A260/280 ratio!) of your plasmid material on the Nanodrop (select dsDNA setting for your plasmid), multiply it by the extinction coefficient (50 ng for DNA), hence:
* conc. (ng/ul) = A260 x 50 - this should correspond to the ng/ul obtained by Nanodrop
Next calculate the molecular weight of the plasmid+insert (total base pairs) and multiply by the average DNA bp mass (~607.4 g/mol), multiply again by 10^9 ng/g to get corresponding ng and divide by Avogadro's constant (6.02 x 10^23 mol), hence:
* mw (ng) = ( total bp x 607.4 g/mol x 10^9 ng/g ) / ( 6.02 x 10^23 mol )
Thus calculate:
* copies/ul = conc. / mw
Now that you know the concentration of your stock, use a small volume to prepare the next highest 1 x 10^x and continue to serial dilute to get the desired copies/ul for your "neat" standard (remember Ci x Vi = Cf x Vf).
Hope this answers your question, let me know otherwise.
Best wishes,
CS
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