Gel electrophoresis and blotting- lecture 14

Question 1

What are the two types of gel used for nucleic acid analysis?

Answer 1

Agarose and polyacrylamide

Question 2

What are the similarities between polyacrylamide and agarose gels?

Answer 2

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.

Question 3

What is the main differences between an agarose gel and a polyacrylamide gel ?

Answer 3

Agarose is good for medium – sized nucleic acid synthesis whereas polyacrylamide if better for small nucleic acids and has a better resolving power

Question 4

How do you prepare polyacrylamide?

Answer 4

oxidation prevents the crosslinking that is needed in polyacrylamide, therefore water is placed on top to prevent this

Question 5

What are the two nucleic acid stains?

Answer 5

Ethidium Bromide and SYBR gold

Question 6

What is an advantage of using an Ethidium bromide stain

Answer 6

Not very safe, but cheap

Question 7

What is the advantage of using SYBR gold?

Answer 7

Safer and stains are better, much more sensitive

Question 8

What are the two stains used to illuminate proteins?

Answer 8

Coomassie blue and silver stain

Question 9

What Is an important choice when choosing electrophoresis conditions?

Answer 9

Must choose whether to have native or denaturing conditions

Question 10

What are denaturing conditions?

Answer 10

Conditions involving chemically treating the nuclei acid or protein samples

Question 11

What does the chemical formamide do to an RNA/DNA structure?

Answer 11

Removes the structure

Question 12

What reducing agent is used to denature proteins?

Answer 12

SDS

Question 13

Why would a denatured gel produce 3 different segments, if a native blot only produces 1?

Answer 13

Denaturation breaks up intra and intermolecular interactions, therefore you get 3 different denatured polypeptide chains

Question 14

What is a Southern blot?

Answer 14

DNA analysis invented by Ed Southern in 1975

Question 15

How is a Southern blot performed?

Answer 15

• 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

Question 16

What is a Northern blot?

Answer 16

Same as a Southern blot, but RNA is analysed instead

Question 17

how do you extract the RNA from the cell?

Answer 17

• Magnetic beads which have T s attached to it

- The poly A tail of the mRNA will bind to the T sequence

Question 18

What is a Western blot used for?

Answer 18

Analysis of proteins

Question 19

what are the differences with a Western blot?

Answer 19

An antibody raised against the protein of interest is used as the probe (as opposed to a radioactively labelled oligonucleotide)

2. Proteins are usually electrophoretically transferred onto the nitrocellulose membrane (as opposed to capillary action transfer)

Question 20

What condition is the Western blot performed under?

Answer 20

denaturing conditions

Question 21

How do we visualize the protein on the gel ?

Answer 21

• protein has 2 antibodies attached to it
• antibody has an enzyme attached.
• when substrate is applied the enzyme converts it into light

Question 22

What is pulse field electrophoresis?

Answer 22

Alternating current. Larger molecules will take longer to change direction

Question 23

What is the point go pulse field electrophoresis?

Answer 23

for larger kilobases

Question 24

What does EMSA stand for?

Answer 24

Electrophoretic Mobility Shift Assay

Question 25

What does EMSA do?

Answer 25

DNA - protein interactions can be analysed . And determine whether a DNA fragment binds to. protein

Question 26

What condition is EMSA under?

Answer 26

Non - Denaturing conditions

Question 27

What does SSCP stand for ?

Answer 27

Single Strand Conformational Polymorphism electrophoresis (also non - denaturing)

Question 28

What can SSCP detect?

Answer 28

DNA mutations

Question 29

How does SSCP work?

Answer 29

• 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