- Take the PCR plate out of the thermocycler when the program is done and centrifuge the plate at 1000 g for 10 seconds. Make sure not to touch the PCR plate with EtBr contaminated gloves, and place it on an adjacent bench so that it does not come in contact with EtBr.
- Plug in the E-Base. Press and release the ‘pwr/prg’ button on the base to select EG (if it is on EP it will still technically work, except your bands will be a lot further down on the E-gel than they normally would be). Generally it will already be set to what you need it to be. Select a run time of 6 minutes by pressing the ‘time’ button.
- Wear blue nitrile gloves because Ethidium Bromide is a mutagen. Remove the E-gel from the package and remove the red plastic comb from the gel.
- Molecular grade water (ddH2O) comes from BIO in plastic bottles, but may have been put in a tube. Pour some water into a tray with a trough in the bottom. Pipette 16 μl of ddH2O (molecular grade deionized water) using an ethidium bromide contaminated 100 multi-channel pipette (it fits 12 tips). Use only the pipettes that are at the EtBr contaminated station. Use 30ul tips. To get water in the control wells (the 13th well marked M) take off 4 tips and turn the pipette so that you fill wells A-G in the 13th row.
- Pipette 4 μl of PCR product into each well using the ethidium bromide contaminated 10 multi-channel pipette. Use 10ul tips. The tips come in boxes that are also labelled with A-G and 1-12 exactly like the PCR plate to help keep track of which row you are on.
- Make sure that the PCR plate is aligned with the E-Gel.
- Change the tips after each row.
- Seal the PCR plate with self-adhering foil and put it in the fridge being careful not to touch the PCR plate with EtBr contaminated gloves.
- Place the E-Gel in the base, the brass contacts on the plate side will fit into the contacts on the board. Press the ‘pwr/prg’ button. The red light will change to green.
- When it is done, the green light will turn red and flash, and the machine will be beep. Press and release the ‘pwr/prg button.
- Throw out any garbage in the garbage container marked “Etidium Bromide Contaminated” (this includes any tips, tip boxes, gloves, used E-gels, Kim Wipes, etc). Anything that has been touched on the EtBr contaminated bench is considered contaminated. PCR plate are not contaminated because they never touch the contaminated bench and they are never handeled with contaminated globes, so consider that non-contaminated.
Next you have to get the E-gel image on the Computer.
Return to PCR Procedure.
Updated April 27 2009