Gel electrophoresis and blotting- lecture 14 Flashcards
What are the two types of gel used for nucleic acid analysis?
Agarose and polyacrylamide
What are the similarities between polyacrylamide and agarose gels?
Both consist of a gel mesh, very tiny holes.
Can place molecule in mesh and apply potential difference across and they will migrate
Small enough holes that they impede the progression via the size of the molecule.
Can separate molecules via size and shape.
What is the main differences between an agarose gel and a polyacrylamide gel ?
Agarose is good for medium – sized nucleic acid synthesis whereas polyacrylamide if better for small nucleic acids and has a better resolving power
How do you prepare polyacrylamide?
oxidation prevents the crosslinking that is needed in polyacrylamide, therefore water is placed on top to prevent this
What are the two nucleic acid stains?
Ethidium Bromide and SYBR gold
What is an advantage of using an Ethidium bromide stain
Not very safe, but cheap
What is the advantage of using SYBR gold?
Safer and stains are better, much more sensitive
What are the two stains used to illuminate proteins?
Coomassie blue and silver stain
What Is an important choice when choosing electrophoresis conditions?
Must choose whether to have native or denaturing conditions
What are denaturing conditions?
Conditions involving chemically treating the nuclei acid or protein samples
What does the chemical formamide do to an RNA/DNA structure?
Removes the structure
What reducing agent is used to denature proteins?
SDS
Why would a denatured gel produce 3 different segments, if a native blot only produces 1?
Denaturation breaks up intra and intermolecular interactions, therefore you get 3 different denatured polypeptide chains
What is a Southern blot?
DNA analysis invented by Ed Southern in 1975
How is a Southern blot performed?
- Gel electrophoresis performed
- ’ blot’ DNA fragments from agarose gel onto the membrane
- membrane imprints with DNA
- Add a labelled probe to the membrane in a buffer solution
- Detection reveals a band where your probe bound to the target sequence
What is a Northern blot?
Same as a Southern blot, but RNA is analysed instead
how do you extract the RNA from the cell?
- Magnetic beads which have T s attached to it
- The poly A tail of the mRNA will bind to the T sequence
What is a Western blot used for?
Analysis of proteins
what are the differences with a Western blot?
An antibody raised against the protein of interest is used as the probe (as opposed to a radioactively labelled oligonucleotide)
- Proteins are usually electrophoretically transferred onto the nitrocellulose membrane (as opposed to capillary action transfer)
What condition is the Western blot performed under?
denaturing conditions
How do we visualize the protein on the gel ?
- protein has 2 antibodies attached to it
- antibody has an enzyme attached.
- when substrate is applied the enzyme converts it into light
What is pulse field electrophoresis?
Alternating current. Larger molecules will take longer to change direction
What is the point go pulse field electrophoresis?
for larger kilobases
What does EMSA stand for?
Electrophoretic Mobility Shift Assay
What does EMSA do?
DNA - protein interactions can be analysed . And determine whether a DNA fragment binds to. protein
What condition is EMSA under?
Non - Denaturing conditions
What does SSCP stand for ?
Single Strand Conformational Polymorphism electrophoresis (also non - denaturing)
What can SSCP detect?
DNA mutations
How does SSCP work?
- get single stranded DNA by cooling on ice
- Due to the mutations, this may affect the folding of the shape of the new structure, therefore the molecules migrate at different rates
