The Cell Biologist Kit

Question 1

What are the 4 approaches of disease modelling?

Answer 1

In vivo, in vitro, ex vivo and in silico.

Question 2

What are the differences between primary cultures and cell lines?

Answer 2

Primary cultures have a finite lifespan, while cell lines can be immortalised.

Question 3

What are the applications of cell cultures?

Answer 3

Used for normal physiology and biochemistry, effects of drugs, mutagenesis and large scale manufacturing of biological compounds.

Question 4

What are the advantages of 3D models?

Answer 4

More similar to in vivo, more realistic biochemical and physiological responses.

Question 5

What are the main principles used in cell cultures?

Answer 5

Aseptic and sterile conditions, growth medium for cells, pH, CO2 and temp.

Question 6

What are the methods used to isolate DNA/RNA and proteins?

Answer 6

Solution based, lyse cells centrifuge collect supernatant and use RNA/DNAase treatment.
Column based, column with diff. affinities.

Question 7

How to measure quality and conc. of DNA/RNA?

Answer 7

Nucleic acids absorb UV light in specific pattern at 260nm.

Question 8

What does the A260/280 ratio show?

Answer 8

It determines the protein contamination of sample. Pure sample should be between 1.8 to 2.0.

Question 9

What does the A260/230 ratio show?

Answer 9

It shows the presence of organic contaminants. Should be around 2.0 for a pure sample.

Question 10

What are the 2 methods used to manipulate genes in vitro?

Answer 10

Small interfering RNA and Short hairpin RNA.

Question 11

What is the process of using siRNA?

Answer 11

2 RNA strands, sense and antisense transferred into the cytosol. Integrated into RISC which separates the 2 strands. Antisense hybridises to the complementary mRNA and cleaves mRNA within RISC. This allow degradation on the target.

Question 12

What is the difference when using shRNA?

Answer 12

shRNA is synthesised within the cell or transfected/tranducted, after which DICER removes the loop.

Question 13

What is Cas9?

Answer 13

Enzyme which has endonuclease activity and can act as a molecular pair of scissors.

Question 14

How does the CRISP/Cas9 system work?

Answer 14

Single stranded RNA guides the Cas9 to the complementary sequence where cleavage takes place. Non homologous end joining DNA repair pathway will attempt to repair cleavage. ERROR prone pathway which will lead to insertions/deletions.

Question 15

What is the process of introducing point mutations?

Answer 15

Denature DNA template and anneal with mutagenic primers containing the mutation. Digest template without the mutation.

Question 16

What is SDS-PAGE?

Answer 16

This will separate proteins based on molecular weight. Proteins are heated with SDS which will make them -vely charged. Move to positive end during gel electrophoresis. Smaller proteins move faster.

Question 17

What are the methods used to detect proteins using specific antibodies?

Answer 17

Western blot, ELISA, Immunohistochemistry and FACS.

Question 18

What is the mechanism of Western blotting?

Answer 18

Isolate the proteins from a sample, do gel electrophoresis, transfer them to a membrane which is blocked with a milk protein and incubate with primary and then secondary antibody.

Question 19

What is the mechanism of Immunohistochemistry?

Answer 19

Primary antibody which binds to antigen, secondary antibody with a substrate which turns brown when the enzyme is added.

Question 20

What is the mechanism of Co-immunoprecipitation?

Answer 20

Add an antibody specific to 1 complex and a protein with coupled beads which can bind to the antibody. This will allow precipitate to form.

Question 21

How is luciferase used for TF activity?

Answer 21

Luciferase bound to promoter with TF. The enzyme with the substrate will create light signal. This will show the promoter activity.

Question 22

What is EMSA?

Answer 22

Electrophoretic mobility shift assay, used for deterring if protein is capable of binding to DNA/RNA.

Question 23

What is Chip-Seq?

Answer 23

Used to determine binding site of DNA associated proteins.