Gel Electrophoresis Flashcards
what is the gel made out of
agarose, when the gel sets at a microscopic level, it is pourous structure which fluids can move through, also mixed with a buffer which contains ions that carry an electric current
how is the gel set up
the gel is poured into an electrophoresis chamber, with wells at the end of one end of the gel. the amplified dna/ all pcr product is pipetted into the wells with loading dye, its then covered with more buffer. the wells at the negative elctrode side of tthe chamber
how does the ddna move through the gel
the dna is placed near the negattive electric charge when connected to s power supply, dna has a negative charge due to its phospahte, the negative charges repel each other causing the dna to move through the gel, away from the negative electrode, towrds tthe positive electrode
how is the dna fragments different sizes
the recognition sites are in many different locations, so is cutt in many differesnt sized fragments
what happens once the elctrophoresis chamber is connected to a power supply
after a while, the gel is stained and photographed
how does thiis sort dna
the shorter fragments of dna move through the gel faster and the longer ones get tangeled up in the matrix of the gel.
what do these lengths of dna show
if a person is homozygous for a gene they will have one line, iif a person is heterozygous they will have two lines in the gel as one allele will be longer than the other
what are surcees of error in gel electrophoresis
sample contamination - wear gloves, gel problems - concentration, comb implantation, sample size, improper loading, electrical current problems - voltage to high/low, plugged wrong way, failed visualisation - too small smaples
what are put into the gels in addition to the dna samples if being used for genetic testing
a standard ladder, a sample of a known healthy gene (negative controol), sample of a mutated gene (positive control)
what is a molecular weight ladder/
sample of dna which have fragments of dna of known molecular size base pairs or kilobasees kb
standards ladder
contaons fragments of known length., fragments of unkown length can then be compared against them to estimate their size
what is placed in the other wells of tthe gel
molecular weight ladder/standard ladder and a negative control which should show no bands in the lane, the gel has likely been contaminated if it does
what factors affect how dna travels
voltage, gel concentration, buffer concentration and time, which is why a standard ladder/ molecular weright ladder is important
what makes the dna visible
it is stained before being pipetted, the most commonn is ethidium bromide which sticks between bases of dna and fluoreses uunder uv light
when is dna profiling used
forensic investigations, mass diasters, identity of human remains and paternity testing
