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