Region molarity and concentration

Question

Hello, 
 I am using the tapestation to analyze a PCR product. When using the region tool, I have extracted between 250 and 1000, as well as between 250 and 5000, but I am getting concentrations, average sizes and region molarities that do not match up. 
 Example for one sample:

From [bp] 
 To [bp] 
 Average Size [bp] 
 Conc. [ng/ul] 
 Region Molarity [nmol/l] 
 % of Total

250 
 5000 
 814 
 44.8 
 133 
 93.15

250 
 1000 
 468 
 36.7 
 127 
 76.46

These numbers look internally consistent (concentration slightly increases, molarity as well) But if you were to calculate the molarity yourself using the average size and given concentration, however, there is a discrepancy for the largest extracted region.

Average Size [bp] 
 Mass weight (dsDNA) 
 Conc. [ng/ul] 
 Conc (nmol/l)

814 
 501411.2 
 44.8 
 89.3478246995679

468 
 288295.96 
 36.7 
 127.299737394863

I don't understand where this discrepancy comes from. I am concerned that the average size is not being calculated correctly, which is the thing I am most interested in here, so that would be troublesome.
Thanks in advance!

Geoff Pettitt · Accepted Answer

Hi, 
 To give you the best possible answer, and so we can work out what is going on in your particular case please could you reach out to your local tech support team and provide the affected data file? By default, data files are saved in Documents>Agilent>TapeStation Data. The extension of the file is named after the assay type. E.g. The DNA D1000 has a .D1000 extension and the High sensitivity RNA assay file would have a .HSRNA extension. You can find your local support team on the contact us page, if you select your country or region from the drop down menu: https://www.agilent.com/en/contact-us/page . 
 I can give you some general guidance on the quantification. Quantification of fragments should be done on the electropherogram view, whereas smears should be analyzed in the region view. The rough algorithm for molarity is as follows: M (Molarity) = W (concentration) x 10 a / (b x MW (size in bp) ) a= 9 (nmol), 12 (pmol), b = 650 (DNA) or 340 (RNA).

Region molarity and concentration

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