Lab 4: Molecular Rainbow Dye Electrophoresis to Identify the - PowerPoint PPT Presentation

Lab 4: Molecular Rainbow Dye Electrophoresis to Identify the Molecules in a Mixture

Notebook Lab Objectives • To understand that molecules vary in size, shape and charge • To analyze the results of molecule separation by gel electrophoresis • To assign relative size of dye molecules as a function of movement through the agarose gel • To determine the components of an unknown dye mixture • To predict how additional molecules would have migrated in gel electrophoresis based upon size and charge

Notebook Gel Electrophoresis Load and Run • Using a p20, load 10 µL of each sample into the gel wells in the following order: (Copy loading order from page 3 of lab) • Run at 125 volts for approximately 30 minutes

What do we know about gel electrophoresis? • Which molecule will migrate down the gel the farthest? 10,000 bp DNA piece 250 bp DNA piece 700 bp DNA piece

What do we know about gel electrophoresis? • Which molecule will migrate down the gel the fastest?

Notebook Results

Notebook TAE Dilution • Prepare _______ mL of 1x TAE buffer to fill the electrophoresis chamber (Include the appropriate formula, calculations and description of how the dilution is prepared in your lab notebook)

Notebook 0.8% Agarose Solution and Gel • Prepare ______ mL of a 0.8% agarose solution to use for gel casting (Show calculations in your lab notebook)

Notebook Dye Loading Order • Lane 1 – Bromophenol Blue (BB) • Lane 2 – Methyl Orange (MO) • Lane 3 – Ponceau G (PG) • Lane 4 – Xylene Cyanol (XC) • Lane 5 – Pyronin Y (PY) • Lane 6 – Unknown 1 • Lane 7 – Unknown 2 • Lane 8 – Unknown 3

Notebook Gel Electrophoresis Load and Run • Using a p20 micropipette, load 10 µl of each sample into the gel wells • Use a fresh tip for each dye sample • Set the voltage on the power supply to 125 volts • Run for 30 minutes • At 5 minutes, record observations:

Notebook Results