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Genomic DNA Fluorescent Example

Document ID: DQ019
Relevant to following products: Manta WorkOut 2.0
Document description: Application Example: Nematodic Worm



A Nematodic worm is transferred to a well of a 384-well plate in a medium containing the fluorescent dye SYTOX Green. A toxic substance is added to each well and with the death of the worms the dye enters the cells, binds to genomic DNA and becomes fluorescent.
 
The analysis must first determine the baseline fluorescence in each single well by calculating the mean value of the first N time points of the measurement. Next calculate at which time the measured fluorescence in each single well exceeds the baseline fluorescence in this well by a specified factor.

This is based around the original idea is published in the paper “Gill et al., Free Radical Biology & Medicine (2003), Vol. 35 pp. 558-565


The instrument is set-up to take kinetic measurements at specified time points.
  1. For the analysis first add a Kinetic Average transform, under the Kinetic Data tab, select Points within specified range only with Min X as 0 and Max X as N (where N is the number of time points to consider).
  2. Then add a Crossing Point transform, under the Crossing Point tab set Crossing Point (Y) expression to w.2 (this refers to the calculated average - i.e. matrix two for each well).
  3. Under the Advanced button select what to do if more than one crossing point is found, for example select Use the lowest. If you want to change the crossing point (the cut-off point) then you can simply modify the expression w.2, for example w.2*1.5 would increase the cut-off point by 50% (and find the point where the well’s fluorescence exceeded the baseline by 50%). Each time you change this setting you can recalculate the results for a new report, or you can create multiple instances of the Crossing Point transform with different expressions to be calculated in parallel.
  4. Now recalculate the results. Under the Analysis tab, the Kinetic Average shows the calculated average and the Crossing Point tab details the results showing the interpolated cycle number where the response crosses the baseline.
If you want to change the range which the baseline is calculated (i.e. N) select the Kinetic Average tab and drag the vertical dashed bars to define a new area, then press calculate. This defined range will be used on all wells.

An example file demonstrating this example is installed within the Results directory and is named “Example Kinetic Average with Cut-Off points.ars”.



Related articles:
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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