BIOC0001 Practical A - Gel electrophoresis

how does DNA move in gel?

with a current, DNA will move towards the positive electrode

without a current, DNA fragments will diffuse outwards in the gel

why does DNA move this way?

DNA is negatively charged, due to negative phosphate groups

what is agarose gel (in context of gel electrophoresis?)

agarose gel is a dense mesh which is made of agarose polymers

presents a physical obstruction to DNA molecules

what are pores in agarose gel?

spaces between the agarose fibres

DNA can move through the pores

what is in the tank for gel electrophoresis

agarose gel with wells (where DNA sample is loaded)

conductive solution (carries charge allowing movement of DNA)

what will happen to longer DNA strands?

will be physically impeded by the agarose gel mesh

will migrate a shorter distance through the mesh

what will happen to smaller/shorter DNA strands?

will be able to move through pores in the gel more easily

will migrate the furthest through the gel

what should be considered when inserting/removing the comb from the gel?

(comb is used to form the wells)

teeth of comb should not touch the bottom of the tank

comb should be removed directly vertically to avoid ripping the gel

what should be considered when adding the buffer to the tank?

ensure there is enough buffer to cover the gel

what should be added to the wells of the agarose gel

DNA samples

DNA ladder/marker (contains DNA fragments of known length for comparison)

where should the electrodes be placed in the tank?

positive electrode at the opposite end of the tank to the wells/DNA samples

what voltage should the gel electrophoresis be run at?

40-180V

what do the different bands mean in gel electrophoresis?

number of bands corresponds to the number of different sized DNA fragments present

why are bands closer together near the top of the gel?

non-linear (logarithmic) nature of the relationship between DNA length and distance

what are restriction enzymes?

represent wide variety of bacterial endonucleases that recognise specific sequences (4-8 bases)

when in the right conditions, restriction enzymes will cleave genetic material at these sequences

how does a restriction enzyme cleave DNA?

hydrolyzes one of the phosphodiester bonds so the DNA chain is broken

when are restriction enzymes used?

frequently used in cloning experiments

use genomic, plasmid and recombinant plasmid DNA

what does loading dye do?

allows the DNA to be visualised