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Quantitative Nucleic Acid (dsDNA, ssDNA, RNA) concentrations for dual measurements (e.g. 260nm/280nm)

Document ID: DQ113
Relevant to following products: Manta WorkOut 2.0
Document description: How To: Setup a protocol for Quantitative Nucleic Acid (dsDNA, ssDNA, RNA) concentrations for dual measurements (e.g. 260nm/280nm)



The protocol described here is used to calculate concentration of Nucleic Acid from dual endpoint measurements (typically 260nm and 280nm). Here the path length correction varies with volume of sample used and also the factor used for determining concentration varies with type of nucleic acid (dsDNA, ssDNA, RNA). With this protocol these parameters can be easily adjusted for different sample volumes and dilution factors for each sample.

The protocol setup is quite lengthy (includes 10 transforms) so an example results file is available for download from here. You can use this as a starting point to make your own protocol or use the following instructions to create your own:

Protocol Setup

Use the Generic Wizard to setup the dual measurements or import and microplate layout. If you use a blank group the wizard will setup blank correction on both raw measurements. Run the protocol and get the raw data into the software.

Press Edit Transforms and add a “Dual Matrix Expression”, use the expression x/y. Next, add an “Expression” transform to store the sample volume, call this Vol and enter the default, e.g 0.2. Next, add another “Expression” transform to calculate the PathLength, use the expression:

#4.Vol/(3.141592654*pow(0.35,2))

Next add two “Matrix Expression” transforms to calculate A for each corrected measurement, for each selected the corrected matrix as the input and use the expression:

x/#5.PathLength

Next add another “Matrix Expression” transforms to calculate the concentration, select the input matrix and enter the expression:

x*50

Return to the main screen and press Recalculate to calculate the results.

Modifying Assay Parameters

To change the volume press Edit Transforms, double click on the first Expression (Calculates Vol = 0.2) and under the Expression tab enter the Vol to use.

To change the dilution factors, press Edit Transforms, double click Dilutions Factors and use the Factors tab to edit the factor by group.

You can also add further transforms as necessary, for example to validate the results or export the data.

You can change the microplate layout as required without affecting the analysis (providing you keep at least 1 blank well).

You can use the Sample IDs button to enter the Sample ID values.



Related articles:
DQ043: How do I create a typical online assay protocol?
How to: Take measurements with an instrument for a typical assay that will be used routinely

DQ032: How do I use the changes I have made to the result analysis in future runs of the assay?
How To: Extract a version of the protocol for use in the future…





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