- Take the E-Gel out of the base. Throw out one glove in the EtBr contaminated garbage. Only one glove can be worn in the hall.
- Hold the E-Gel with your gloved hand.
- Go in the dark room and open the door of the cabinet beside the computer, and put the E-Gel inside. There is a box propping open the door because the light will turn on if it is not open, remove the box and remember to place it back when you are finished.
- Open the TEST icon on the desktop.
- Login: HANNER , Password: bob (make sure that CapsLock is off for bob because it is case sensitive).
- Place your E-gel in the cabinet, leaving the door open.
- On the computer screen, press the “Aquire” box at the top left.
- Adjust the E-gel position, zoom and focus so that you can see the crosshairs.
- Close the door.
- Set the exposure to 2 seconds, UV transilluminator, filter position to 2, and press “Expose Preview.” The image should now appear on the screen.
- Press “Aquire Image.”
- Adjust the “white” and “gamma” levels as needed to get a clear image. Remember that it always prints lighter than you see on the screen. Print a copy of the picture.
- Brighten the image so that it is lighter before selecting “Save modified.” Save the image as a jpeg.
- 4. Placed the saved image into the proper folder within My Documents and copy the image onto a USB key (you will need the image later to upload to LIMS). When naming the file, name it after the box name/number, and the PCR primer that you used (eg. HIC-002_LepR_MlepF).
- 5. The E-Gel can be used once more. Label the bag the E-gel came in with your name, the date, and the box number. Make sure to put the red plastic comb back in the E-gel, put the E-gel in its labelled bag, and put that into a labelled sealed bag on the ethidium bromide contaminated bench.
You are looking for distinct white bands that show up in the middle, between rows. If you get bright blobs of white that seems to have moved farther than your distinct bands, these are primer-dimers. Primer-dimers are when primers stick together rather than onto DNA to amplify it. That happens when your PCR has failed.
You need to have at least 85 bands that are visible to be able to have the plate sequenced. If you have less than 85 bands, then you can hitpick the plate along with another that was also partially successful, which will take out the samples that amplified, or the DNA could be used with a different primer to see if it works better. See Getting Plates Hit Picked and Dataserv. If you have something very urgent you might be able to get away with using less than 85 bands. You could also fill a non-skirted plate with the quantity of Seq Mix and primer that you need, and tell BIO that the plate is not full – this is not recommended. If you have problems, see What to do when your Plates Fail.
Make sure to put your E-gel image into LIMS.
Updated April 27 2009