Download Gel Electrophoresis

Gel Electrophoresis
Biotech I
Gel Electrophoresis
• Definition: the process of separating
molecules based on size and charge
• Agarose: highly purified agar, heated and
dissolved in buffer. Forms a matrix of
pores for molecules to travel through.
– Smaller molecules travel further
– Molecules migrate towards the
– positive (red) end of the chamber
Gel Electrophoresis
• Process
– Make Agarose gel
• Thinner gels (0.8%) yield better results for larger DNA
– Prepare samples
• Restriction enzymes used to cleave at specified sites
– Apply samples to gels, apply current
• If samples run from positive end they will run off the gel
– Stain gels to see bands
• Would not be able to see bands if we did not stain
Gel Electrophoresis
• DNA molecules have a negative charge
– This allows them to migrate towards the positive end of the
chamber
• The samples and the electrophoresis chamber use
specialized buffers. Using
• TAE/TBE buffer helps stabilize the sample
• and allows the reaction to occur quicker in
• the chamber.
– If water were in the chamber instead of TAE/TBE buffer the
reaction would take much longer or migration may not occur at
all
• Stains: ethidium bromide will cause the bands to glow
orange under UV light. Fast stain will result in blue
bands
Uses for Gel Electrophoresis
• DNA fingerprinting or profiling
– Paternity testing
– Crime scene sample analysis
– Identification of bacteria and other pathogens
• Who is credited with discovering the DNA
profiling process?
– Alec Jefferies in 1985