AlphaView user guide

This is a brief tutorial on how to use the AlphaView software that is available on the Virtual Campus Website. This tutorial will help you go through the analysis steps you need to do to determine the optimal amount of enzyme to use in your assays next week. There are many other free programs that can do similar analysis (such as ImageJ), so you are not locked into using this program. 1) Install and open the AlphaView software. 2) Open the figure you want to analyse. a. Select the File menu. b. Then, click on Open. c. Choose a picture with a valid format (.tif).

3) Your picture should be fairly straight. You can always rotate your picture by using the rotation tool in the left panel of the screen (Rotate-Flip). You can also slide the Black and White tabs on the left of the screen to adjust the intensity of your figure (do not oversaturate your bands).

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4) In some situations, you will need to crop out one series of reactions to analyze. To do this, select Edit Activation under the Edit menu. You can then highlight the section to analyze and click Crop. Make sure you get everything you need in the cropped image. Important Note: There is no undo function in this program. Save frequently and think twice before finalizing a command.

5) To analyse the intensity of our DNA bands, you need to select the Analysis tools tab. Select the Lane profile and then Lane tab. If you scroll down you can see there is a Number of lanes function. Make sure this is set to the right number that you need to analysis. The computer program will now draw some lanes on the gel. Unfortunately, they do not line up with our results; however you can stretch the lanes to fit your gel. (see figures on the following page) You cannot edit the lanes separately with this function (you want to keep everything the same for each lane so this is not a problem).

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6) Once the lanes fit your sample. You can now click on Detect bands. The program assumes that the bands are lighter than the background. You can change this by selecting the Bands darker than background option if your picture was inverted (white background and black DNA bands). 7) Here the program has detected bands for our gel. The bands are numbered based on their order from the top of the gel. The program also displays a histogram with the peak intensity for each band detected in the selected lane (Lane 1 in this example). Note that two bands visible in Lane 1 were not detected by the software and does not have an intensity associated with them (Red arrows).

8) We can fix this by adding bands. In the Band tab there is an option for adding/deleting bands. Select the Add band function and click on the band you want to analyse. 9) Once all the ladder bands are selected. You can do the same for the remaining lanes. If a visible bands is not selected in a specific lane. Select the lane by clicking on it. Then, select the Add band option and click on the band you want to select (see Lane #2 and 3 on the following pages picture).

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The real strength of programs like Alphaview is that they can generate standard curves and calculate densitometry for us. The next few steps will go through this process. 10) Click on the MW tab in Lane profiles. Click on the Add button and click on any ladder band (Its best to start at the top). Now enter in its MW in bp; repeat this for each band visible.

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11) The program assumes that the MW migrations are logarithmically related and will generate a standard curve with R2 values as you add MW markers. If your curve isnt great, you can remove outlier bands using the Remove Band button under the Band tab). As you can see on my example, the uppermost band of my marker seems to be an outlier. Let see what will happen if I delete that band (red arrow).

12) This modification improves the curve (R2 of 0.9165 instead of 0.8452), but note that all the other bands have been renumbered because they start from the top. It is important to keep this in mind. You need to keep a copy of this ordering so you remember which band #s in your final report relate to which band on the gel. 13) We can do a similar standard curve generation using masses. Click on the Mass tab and make sure that the option is set to Mass standards. This assumes each band has a known mass. The other option Molecular Ladder assumes that you know the total mass of your ladder, but not of each individual band. Click on Add and insert a mass for each individual band of the marker (see following picture).

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14) Now we can export our data. Select the Report tab and make sure the Data Sheet and Graph functions are selected. You should also click on the Full image option (rather than or sized) or the image may be too small to see. Click on the Next button to generate the report. Then press on the Generate report option.

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15) First section of your report contains an image of your gel with the bands labelled with their band numbers and any standard curves you generated.

16) The second section is where you will find all the calculated MWs and masses for your bands. Because of the numbering system, the same band in different lanes may have different numbers. Again, this is why you need a copy of the gel image. The green section corresponds to your DNA marker. 17) You can save the report as an HTML file. You can also copy your mass values into excel to test the linearity of the data (see picture on following page). It will be particularly useful when you will need to figure out the best enzyme amount to use in Lab#8.

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18) You are now ready to analyse your data. Using excel, you can analyze this data in three different ways, with increasing degrees of complexity, and plotted the data. a. The first is the raw intensity (mass) of the major band present in reach reaction plotted vs time.

180 160 140 Intensity 120 100 80 60 40 20 0 0 5

Intensity vs time

R = 0.9751

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15 Assay Time

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b. The second is the total intensity of enzymatic products for each time point added together and then plotted vs time. If you look carefully at my example, you will notice that in lane 7, a second enzymatic product with a higher molecular weight than the original band becomes visible. If it happens to you, you can add the intensity of both product bands. It will correspond to the total intensity of enzymatic products. 9 James Butcher (2011)

c. The third normalizes the total intensity of enzymatic products for each time point added together, to your loading efficiency in each well. Im not going to tell you how to do this normalization. There is a way to tell your loading efficiency across each lane in your enzymatic reactions due to a factor that should not change across conditions.

5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0

Normalized Total Intensity vs time

Intensity

R = 0.9778

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