The intention of this video is to answer some frequently asked questions.. This segment of the video discusses loss of resolution, including the significance of Full Width Half Maximum (FWHM).
How to differentiate between loss of resolution from amplification issues and capillary quality in samples that do not contain internal control probes?
Loss of Resolution FWHM
Full Width Half Maximum (FWHM)
-1 or less indicates single base pair resolution
In Amplicons may be from:
-Amplification or DNA quality factors
-Excess salt
-Failing capillary
In Size Standards:
Excess salt
Failing capillary
-The screenshot below shows the main screen of GeneMarker HID v3.0.0 and there’s a FWHM column circled in red.
-The top example (ref-1-2.hid) has lost that single base pair resolution and the bottom example (ref-1-3.hid) is within single base pair resolution.
FWHM of Size Standard
-Loss of resolution in the internal lane standard
-Indicates loss of resolution is not from amplification
-Evaluate potential causes and repeat the electrophoresis
-Below are the same samples as shown in the above screenshot, but now looking at the size standard dye channel.
-The sample (ref-1-2.hid) that had the loss of resolution in the last screenshot, shows the same loss of resolution in the size standard.
-Since the size standard does not go through DNA extraction or amplification, like the amplicons, this would indicate either excess salt or capillary failure for the reason of loss of resolution in this sample.
-The sample (ref-1-3.hid) shows good FWHM and resolution in size standard like it did in the amplicons shown in the previous screenshot.