PicoGreen Overview

Overview of PicoGreen

Preparation:

• Take the Picogreen dye out of the fridge and allow it to come to room temperature to thaw. 
• Check to be sure there is enough TE for the procedure. TE can be made by adding 500 µl of 0.5M EDTA to a full bottle of EB (Elution Buffer). 
• The fluorescent plate is usually found to the left of the sink in the robot room, locate one of these for each Picogreen reaction you perform. 
• You will need your plate labeled "(Plate ID) PCR Frags (Today's Date)", reservoir with water, reservoir with picogreen, and fluorescent plate. 
• 25mL of TE are mixed with 125 µl of picogreen dye for every two plates processed. BE SURE TO USE THIS MIXTURE IMMEDIATELY. It is light sensitive-the longer it sits out in the light the more your results will be affected.

Procedure:

• PicoG1 dilutes a portion of the PCR fragments with water then mixes that dilution with the picogreen dye before adding the entire mixture to the fluorescent plate. 
• Select the FluoroStar program on the Robot computer. Highlight "User" and click "ok". At the bottom left of the screen is a box that reads either "Fluorescence" or "Absorbance". By clicking on that box you can change the function. Be sure it reads "Fluorescence" for the Picogreen procedure. 
• Open the top of the FluoroStar machine and roatate the cords so the Fluorescence cords are at the top. These are the silver cords. 
• Back at the computer, click on "Picogreen Assay". Label your experiment run with your plate id and the date. Click "Run Test Sample". 
• Your results are put into an Excel spreadsheet. You can access this spreadsheet by opening FluoroStar and selecting the "X" -with a spreadsheet behind it- at the top of the screen in the middle of the tool bar. 
• Look for direction on how to access your results in the protocol "FluoroStar Results".
  
  
• Next Step: Manual LIC setup.