Dr. Zeytuni Methods

Question 1

what are the 2 main general ways of studying protein function?

Answer 1

gain or loss of function

Question 2

what is PCR used for?

Answer 2

amplify cDNA of gene f interest

Question 3

what are the 3 steps of PCR

Answer 3

denaturation, annealing, extension

Question 4

how many clones of your DNA segments do you haveafter 1, 2, 3 pcr cycles?

Answer 4

1 cycle = 2 clones of DS DNA
2 cycles = 4 clones
3 cycles = 8 clones

Question 5

after pcr, whats the next step when overexpression a protein?

Answer 5

cloning the PCR product into a plasmid vector

Question 6

how is a gene introduced in a vector?

Answer 6

restriction enzymes cut vector -> annealing of your gene -> covalent linkage by DNA ligase

Question 7

why is there ana antibiotic resistance sequence in a vector?

Answer 7

to select the bacteria that contain the plasmid only; the others will die

Question 8

then, what do you do with your plasmid vector?

Answer 8

introduce in bacteria: heat shock to permeabilize the membrane -> cell culture

Question 9

3 ways to extract your plasmid from bacteria after culture

Answer 9

• Basic solution (NaOH): gentle extraction of the plasmid
  without chromosomal bacterial DNA
• Phenol/chloroform extraction: isolation of the plasmid,
  get rid of the proteins
• Ethanol precipitation: purified plasmid prep

Question 10

how can you transfect plasmid DNA in eukaryotic cells?

Answer 10

via liposomes

Question 11

what are the advantages of expressing your protein of interest in E. Coli?

Answer 11

• Produce large amounts of purified protein
• Easy to extract from bacteria
• Easy to purify
• Useful to produce specific antibodies raised against your protein of interest

Question 12

how do you purifiy your protein from bacteria like E. coli?

Answer 12

by affinity chromatography using
Glutathione agarose beads

Question 13

to purify your protein with gluthathione agarose beads after its replication, what does the plasmid vector need to have?

Answer 13

GST (glutathione-S-Transferase) gene

Question 14

how do you produce monoclonal antibodies?

Answer 14

1. inject mouse with antigen
2. mouse creates antibodies
3. isolate immune cells (antibody forming cells)
4. fuse with a tumor cell
5. hybridomas produces antibodies

Question 15

what are differences of making monoclonal vs polyclonal antibodies?

Answer 15

ply: make in rabbit
- antibody recognizes multiple epitopes
- Limitation of availability of
antibodies

Question 16

how does western blot work lol

Answer 16

SDS-PAGE membrane transferred into a nitrocellulose filter -> incubated with a primary antibody against protein of interest -> incubate with a secondary antibody linked to a radioactive probe or HRP -> detection

Question 17

what is epitope tagging and what is it used for?

Answer 17

• tagging POI with an epitope
• allows purification and localization of POI via a commercial antibody

Question 18

why can’t you always use epitope tag?

Answer 18

they sometimes interfere with the function

Question 19

why can’t you always do loss of function experiment?

Answer 19

sometimes the protein is essential

Question 20

how does siRNA work?

Answer 20

RISC complex cleaves the RNA, allowing for cleavage of the dsRNA and introduction of the siRNA

Question 21

3 ways of gene editing?

Answer 21

1) gene replacement
2) gene deletion (knockout)
3) gene addition (adding a mutant and see if it overrides the function)

Question 22

explain CRSPR CAS9

Answer 22

single guide RNA allows for Cas9 endonuclease to cut the target DNA