Gel electrophoresis Flashcards
What are the steps of gel electrophoresis?
- Cut up sample DNA with restriction enzymes
- Put sample in a well of agerose gel
- “Run” the gel by running an electric current through it
- Use a UV light or equivalent to analyse the banding patterns
Will larger or smaller fragments of DNA move further?
smaller fragments will have less resistance therefore will move further from the wells.
large fragments will not move far due to higher resistance
What charge does DNA have?
Negative (therefore attracted to the positive terminal)
What is a well?
an indent in the gel into which a DNA sample is loaded
What is agerose gel?
a sponge-like gel used in gel electrophoresis that contains pores for DNA fragments to move through
What is a standard ladder?
a mixture of DNA fragments of known length that are used to infer the size of fragments in a sample
What is a buffer?
an ion-rich solution that carries electrical current through the agarose gel
What is a band?
a line seen in the gel after running gel electrophoresis that corresponds to a collection of DNA fragments of a specific size
What is ethidium bromide?
a fluorescent dye that binds to DNA fragments in a gel and allows them to be easily visualised under ultraviolet light
What is an electrode?
conductors of electricity that are attached to both ends of a gel allowing an electrical current to pass through it
Does DNA of the same size always travel the same distance?
No
Why does the distance DNA fragments of the same size travel in gel electrophoresis differ?
voltage
gel composition
buffer concentration
time
How does voltage affect gel electrophoresis?
the stronger the electric force generated by the electrodes the further DNA travels towards the positive electrode
How does gel composition affect gel electrophoresis?
gels with a greater density and agarose concentration increase the difficulty for larger fragments to move through
How does buffer concentration affect gel electrophoresis?
the greater the concentration of ions in the buffer the more the electric current is conducted through the gel, which causes DNA to move further down the lane
How does time affect gel electrophoresis?
the longer the electric current is applied, the further the DNA will travel. Note: if too much time passes, the DNA may move out of the gel.
What is a short tandem repeat?
STRs are small sections of repeated nucleotides that vary in length between people and are found in the non-coding areas of autosomal chromosomes
What can STRs be used for?
DNA profiling
Why is 2% agerose gel better for gel eletrophoresis than 1% agerose gel?
smaller gaps
bigger DNA blocked
smaller DNA can pass
