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

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

Notebook

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?

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

Notebook