Frequently Asked Questions
How can I ensure the quantitative accuracy of my HCP coverage analysis using SpotMap?
In this video we show you how automatic spot outlines in SpotMap obtain an objective, accurate and quantitative analysis and how you can use 3D mode to validate the software you currently use for HCP coverage.
How do I use Image QC in SpotMap?
Image QC is used to quality check any images added to your project, before you run analysis. This helps to save time as any issues with the image are immediately highlighted, for you to address before any further analysis is attempted.
Image QC flags any potential issues with:
- Image file size
- Manual editing of the images
Poor image quality can make analysis of your gels and blots difficult and may reduce the objectivity of your analysis.
How can I purchase the software?
Our software is available directly. It is also available with a number of gel documentation systems. Please contact us at email@example.com to get more details.
I have further questions. Can I have a product demonstration?
Can I install CLIQS on a machine with no internet connection?
Yes you can. It is called a manual installation and if there is no internet connection on registration then you follow the instructions provided to get a license working. If you plan to use the machine offline then make sure it is unplugged on registration.
I haven’t had a chance to use the trial can I have an extension?
What Operating Systems can I use?
All of the latest TotalLab products will work under Windows 7 and Windows 8 and Windows 10, 32 and 64 bit versions.
Why does automatic lane creation fail on my gel?
There could be several reasons why this happens…
- The lanes are short or have very few bands
- The lanes are too close together so the algorithm cannot split them
- The lanes are not quite the same width which is an assumption the algorithm uses
- The resolution of the image is very low and there is not enough band information
Using an AOI for lane detection can help (draw a box when in automatic detection mode) or simply try to create the lanes manually.
You can also contact us with any support questions.
How does Molecular Sizing work?
Each lane has its own calibration curve calculated from where the iso-molecular weight lines (yellow) cross the centre of a lane.
With only one standard lane these curves are usually the same as the lines just run horizontally across the gel.
However, with more than one standard lane it joins up the bands of the same value and so the lines can be slightly off horizontal depending on the gel.
Why use different curve types?
The default curve fit is cubic spline.
This curve type creates a smooth curve passing through all points, it has no defining equation.
This option can be somewhat over sensitive to inaccurate mappings but will guarantee that the results are as expected from where the iso-molecular weight lines (yellow) cross the lanes. First order Lagrange is similar but uses straight lines.
The other main option used historically is the Linear Log equation. This is a straight line calculated from the log of the Molecular Size. This is theoretically correct but can lead to misleading results due to any inaccuracy of the curve fit. Even curves with high R2 results can still have problems.
What is the name of the curve fitting algorithm?
We use an iterative algorithm called Levenberg-Marquardt to get the results for the curve fitting equations. Cubic Spline and First order Lagrange are also well defined algorithms.
My image does not look like the image I captured. Why?
A histogram of all pixel values is created. Then the default auto contrast is created from the value of the histogram after 0.5% of all pixels and at 99.5% of all pixels. This stops outliers or scanner noise affecting the result but it can look a little different
If the image has a calibration (from Powerscan) or an encoding (from GE or Fuji scanners) then the converted values are used by default for the image. If you do not want this then use the “Raw” option to go back to the raw pixels values
Note: Contrast does not affect the values used in any calculations.
My profile is upside down. What can I do to fix it?
Go to the File menu and select Invert Measurements to reverse the calculation used to generate the Profile.
This option is remembered so you should not need to change it again.
When I load in my JPG file the image is inverted.
This occasionally happens as part of the loading function.
Just press the invert button on the toolbar to see if reversed again.
I cannot load my tiff image?
This may be because we can’t load multi-page tiff.
There are many free tools which can split the tiff into single image files including ImageJ and ImageMagick.
The following file formats are supported by our CLIQS software:
What is Band Volume?
This is defined as the total value of all the pixels in a band with background subtracted. The pixels are shown on the image as between the 2 red lines (the band edges).
Another way to look at it is as the area under the profile for the band (with background subtracted) multiplied by the lane width.
Where can you get the total volume of all the bands in a Lane?
This information can be found in the Analysis Report (previously called Gel Report). On the first page total band volume (and total lane volume) are displayed in a summary table of all the lanes in the gel.
What is the background level in Array and why don’t all spots have the same background volume?
The background level is the value calculated from the selected method as the background used for that spot. It can be the same for all spots.
The value is subtracted from every pixel in the spot and then all pixels are summed to get the spot volume. If some pixels in the spot are lower than the background level then they are only set to zero and not a negative number. Therefore you can get differing background totals, though not by that much usually.
What is Profile Deconvolution?
This option allows the user to model each band as a gaussian and use these to recalculate the band volume. This is sometimes used to split the volume of overlapping bands in what is believed to be a more accurate manner.
Advanced fitting is best but there are some draw backs. Firstly, this can be very slow and secondly, without good background subtraction they can be very wrong.
How do I turn Profile Deconvolution on?
By default in CLIQS this option is off. You can turn the option on from the start or finish pages of the wizard by ticking the check box next to Profile Deconvolution.