Appendix G

APPENDIX G. Instructions for Capturing Digital Images of Nucleic Acid Gels Stained with SYBR Safe Using the Kodak Gel Logic 200 Imaging System

SAFETY WARNING: The UV lamp should automatically turn off, when the imager door is opened. However, as a safety precaution, you should wear protective goggles. You should also wear protective gloves against chemical contaminants such as ethidium bromide.

1. Open the door of the gel imager. Make sure the white light pad is not on the imaging area. If it is, remove it and place it in the cabinet below the imager.

2. Remove your gel from its container and place it directly on the glass. Center it on the imaging area.

3. Close the door of the imager and turn the dial to TRANS UV.

4. Open the Kodak 1D 3.6 application from the desktop.

5. Click on the “Capture GL 200” button at the top left.

6. In the Capture (Gel Logic 200) window, select “SYBR Green” from the “Custom Settings” menu.

7. Click on the “Preview” button at the top right. The camera will begin taking live images of the gel.

8. If the gel image is too big or too small, manually adjust the size at the camera level (the middle ring). A setting of about 30mm works well.

9. Focus adjustment is also done manually at the camera level (bottom ring). Set at approximately 5mm. There is only one focusing ring (no separate coarse and fine controls). NOTE: The “coarse” and “fine” adjust buttons on the computer screen control exposure time (not focus).

10. Using the top ring of the camera (above the UV light chamber), open the lens aperture to its maximum. Due to the increased light intensity, the bright bands will be saturated. However, this will allow you to see the weakest bands. Now set the aperture to the desired opening size (f-stop) for best overall image appearance (usually, around 2–4).

11. Fine-focus the image using the bottom ring of the camera lens. NOTE: The middle ring is a zoom control for changing the magnification of the image.

12. For BISC 110 nucleic acid gels, an exposure time (shutter speed) of 1.0sec is a good starting point. If you increase exposure time and the gel turns red, that means the signal is saturated. Use the “fine” adjust button to decrease the exposure time, until the red disappears. Alternatively, if there are some very faint bands on the gel which you need to see, even at the expense of brighter bands being overexposed, you may want to uncheck the “Show Saturation” box, so that the red color is not displayed.

13. When you have finished adjusting your image, click on the “Capture” button (below “Preview”). A snapshot of your gel will now appear in another window called “NewProject 1*.” An “Image Display” window also appears at this time. You can change the brightness/contrast here using the “image histogram.” Under the “Display” menu, you can invert the image to save printer toner or ink.

14. To save your data: Select File > Export Data > Image. From the “Save as type” menu, select JPEG and save your image to the desktop. This computer is networked to the SCI 314 printer, so you can print out a copy of the gel image. You should also transfer your file to your FirstClass account, or place it directly into your BISC 110 lab conference. Please do not leave un-filed images on the desktop for an extended period.

15. Close the NewProject 1* window. Go to step 5 of the instructions for the next gel.