day 4 Flashcards
(13 cards)
what is the purpose of gel electrophoresis
allows us to analyze different conformers of plasmid DNA
assess if ligation worked
determine the # of cut sites in the plasmid
what is the purpose of comparing digestion patterns in plasmids
sequence differences in cut sites may indicate that the PCR product came from a different species
how does agarose work? what is the purpose of the electric current
the agarose creates a matrix that allows nucleic acids to pass through the pores when in the presence of a electric current
negatively charged molecules will travel towards the positive node
if you want to separate larger fragments of DNA, what concentration of agarose should you use?
smaller concentrations create larger pores which are better for separating large DNA fragments
if you want to separate smaller fragments of DNA, what concentration of agarose should you use?
higher concentrations create smaller pores which are better for separating smaller DNA fragments
what is the purpose of the loading dye?
it has a blue dye that allows us to visualize the progress of separation but it also contains glycerol which is denser than the buffer and allows the samples to sink into the wells
what is the purpose of GelRed dye
the gel red dye binds/ intercalates with the bases in DNA and it emits a red fluorescence when exposed to UV light. This allows us to visualize our samples
what are the two factors that DNA migration in electrophoresis depend on?
the size/length of the fragments (e.g, larger fragments with more base pairs will move through the gel more slowly)
the conformation of the fragments (e.g, tightly coiled fragments will move faster than loose fragments)
what is the supercoiled confirmation of the plasmid? how fast does it migrate
supercoils are formed by enzymes like topoisomerase which cause it to form compact balls
this conformation migrates the fastest
what is the open circle conformation of the plasmid? how fast does it migrate
the open circle conformation is caused by a small nick in a phosphodiester bond, where only one of the strands breaks. This causes a loose open form of DNA that moves slower than linear and supercoiled DNA
what is the linear conformation?
it is when both strands have phosphodiester breakages in their backbones, this causes the DNA to linearize
it moves faster than the open-circle conformation but slower than the supercoiled
what are catenanes? how fast do they migrate?
They are replicated plasmids that have not yet separated and remain as linked circles
they migrate much more slowly than all other forms of DNA since they contain two plasmids linked
what is the order of migration based on conformation
supercoil> linear > circular > catenanes
