Software Requirements

ImageJ (optional):	Download
GelAnalyzer 2010a:	Download

Convert TIFF -> PNG

Skip to Detect Lanes if your gel image is already a high-resolution PNG-formatted file, with black bands on a white background.

1. Open ImageJ software, then drag/drop your gel TIFF onto the ImageJ menu

2. Convert the TIFF to 32-bit black and white by selecting Image -> Type -> 32-bit

3. Invert black and white by selecting Edit -> Invert

4. Save image as PNG by selecting File -> Save As -> PNG...

5. Enter a unique filename for your PNG, such as 1-1-32bit.png, then select Save

Detect Lanes

1. Open GelAnalyzer 2010a and select the New Analysis button

2. Move to the folder containing your saved PNG file from the steps above, then select the saved PNG file and select Open

3. Select the image type by checking the Dark on light radio button, since the bands are dark and the background is white, then select Ok

4. Select the Lanes mode button

5. Select the Detect lanes button

6. Verify that all lanes have a box around them, as shown below.

NOTE: If too many boxes are drawn, a box is improperly drawn or if any lane(s) is missing a box around it, you may have to manually edit the gel.To manually edit the gel, please refer to the following sections, otherwise continue to the next section:

• Modify box around lane
• Add box around lane
• Delete box around lane

Detect Bands

1. Select the Bands mode button

2. Select Detect bands on every lane button

3. Verify all relevant bands have a box around them

NOTE: If you notice that a band(s) are missing a box, then you should add a box around the band

Delete Bands

1. Select the lane with extra band(s), then in the Profile window right-click on the peak you would like to delete

2. Click OK to delete the band

3. Verify the band has been removed

Calculate Volumes

1. Select the Background subtract mode button

2. Select the Rolling ball button

3. Set the Ball radius to 100, then select Ok

4. Select the All lanes radio button, then select Ok

5. In the Analysis info window, you will notice the band values. To show the values for all lanes, select the Show -> All lanes radio button

6. With all the band values for all lanes shown, select all values from the Analysis info window using copy (Ctrl + c), then paste (Ctrl + v) into the calculator below

Calculate Gene Editing Percentage

NOTE: Band1 is considered the uncut control band, while bands 2-X are the fragment bands. All bands in the table are used for calculating the percent gene editing, so if you want specific bands excluded from the calculation, make sure they are not in the Analysis info table (i.e. loading well band or run-off band).

How to calculate % gene editing:

Add Lane

1. Select the Lanes mode button

2. Select the Add new lane button

3. Left-click the cross-hair in the top-middle of the lane of interest

4. Drag the cross-hairs to the middle-bottom of the lane

5. Left-click and drag the cross-hairs to the left or right until lane is boxed in

6. Left-click the cross-hairs to complete the box

Go back to previous section

Delete Lane

1. Select the Lanes mode button

2. Select the lane to delete (left-click on lane), then select the Delete selected lane button

Go back to previous section

Modify Lane

1. Select the Lanes mode button

2. Select the Modify lanes button

3. You can resize the height of the box or adjust the curve of the box by left-clicking and dragging the yellow filled-in box at the top of the lane

4. You can adjust the width of the lane by left-clicking and dragging on the left or right side of the box around the lane

Go back to previous section

Add Band

You may notice that the automatic band detection misses a band(s), so you may have to manually add a box around the band

1. Select the Bands mode button

2. Select the Add new band button

3. Move the cross-hairs over the Profile image and left-click on the left side of the peak of interest

4. Move the cross-hairs over the Profile image and left-click on the right side of the peak of interest

5. Verify the box was drawn around the correct band and verify that a new label was added to the Profile image

Go back to previous section