Labs

Question 1

What are the differnet micropipette volumes?

Answer 1

P10

P20

P200

P1000

Question 2

how do you balance a centrifuge?

Answer 2

ensure eppendorfs full w equal volumes at opposite points to each other- if only one then use water in a ‘blank’

Question 3

what is molarity?

Answer 3

measure of concentration of solute in solution

Question 4

what are moles?

Answer 4

molecular weight of a substance expressed in grams

Question 5

molarity = ?

Answer 5

moles per litre

Question 6

distinguish between moles and molarity

Answer 6

moles- quantity of material

molarity- concentration of material in solution

Question 7

what is avagadros?

Answer 7

one mole of substance

Question 8

what calculation is used for stock solutions?

Answer 8

C1V1= C2V2

Question 9

what is a lowry protein assay?

Answer 9

determine protein concentration in unknown sample

Question 10

what reagent is used for colour chnage in lowry?

what is the colour change?

Answer 10

folins reagent

yellow/brown to blue

Question 11

what is used to measure absorbance of samples in protein assays?

Answer 11

spectrophotometer

Question 12

describe in vitro/ in vivo

Answer 12

in vitro- in glass

in vivo- in life

Question 13

which cell type was used in tissue culture?

Answer 13

COS7 cells

Fibroblastic cells from monkey kidney

Question 14

what terms can be used to describe splitting cells in culture?

Answer 14

sub culture or passage

Question 15

what is passaging?

Answer 15

removal of media and transfer of proportion of cells into fresh gorwth medium

Question 16

what are the growth phases of cell during tissue culture?

Answer 16

lag phase

log phase- proliferation of cells

Question 17

how often were the cells cultured?

Answer 17

twice a week

Mon/Fri

Question 18

what is PBS?

Answer 18

Phosphate Buffered Saline

used to wash the cells

Question 19

what enzyme was used to detach the cells from the bottom of the culture dishes?

Answer 19

Trypsin EDTA

Question 20

What growth medium was used in tissue culture?

Answer 20

DMEM media

Question 21

what were the components of the DMEM?

Answer 21

10% FBS (extra nutrients)

glutamine- essential amino acid

sodium pyruvate- carbon source

1% penstrep- prevent infection

phenyl red dye- pH indicator

Question 22

what is confluency?

what % is best when passaging in culture?

Answer 22

coverage of dish by cells

80%- as they are in the log phase of growth at this point

Question 23

What in the DMEM media inhibits trypsin activty?

Answer 23

calcium

thsi is why cells are washed with PBS

Question 24

describe a 1:5 split

Answer 24

2ml cell suspension into new dish + 8ml DMEM

Question 25

What is GFP?

Answer 25

Green Flourescent Protein

Question 26

What bacteria were used for culture?

Answer 26

E.coli

Question 27

what was the broth that the bacterial colony was transferred into?

Answer 27

LB broth containing candymiycin

Question 28

what is a plasmid?

Answer 28

double stranded, circular extra chomosomal DNA of bacterium

Question 29

what method was used to isolate the plasmid DNA?

Answer 29

alkaline lysis

Question 30

describe isolation of plasmid

Answer 30

cell growth and harvest re-suspension (TRIS, EDTA, Glucose) lysis (SDS, NaOH) neutralisation (potassium acetate) cleanign and concentration (ethanol)

Question 31

what are the components of alkaline lysis buffer?

Answer 31

SDS detergent- cleaves phospholipid bilayer of membrane Alkali- NaOH, denatures structural proteins of cell membrane and breaks hydrogen bonds

Question 32

what was in the suspension solution for plasmid DNA?

Answer 32

TRIS- buffer Glucose- osmotic pressure EDTA- chelates divalent cations Mg+, Ca+

Question 33

what does the potassium acetate do during alkaline lysis?

Answer 33

neutralises solution allowing hydrogen bonds between bases to re-establish

Question 34

why is addition of potassium acetate important?

Answer 34

selective process- plasmid DNA can renature but gDNA is too big (make sure not to vortex as this can shear gDNA into smaller bits that can reaneal)

Question 35

how is double stranded plasmid DNA separated from gDNA, SDS and denatured proteins?

Answer 35

dissolve easily in soln whilst others stick together to form precipitate seperarated by centrifugation

Question 36

what is used to seperate the DNA?

Answer 36

isopropanol

Question 37

how is plasmid DNA cleaned (debris, EDTA, salt)?

Answer 37

ethanol precipitation

Question 38

what is transfection?

Answer 38

introdunction of foreign DNA into nucleus of eukaryotic cells

Question 39

what are the two types of transfectant?

Answer 39

stable- cells have intregratde foreign DNA Transient- foreign DNA does not integrate in genome but genes are expressed for limited time (24-96hrs)

Question 40

what methods can be used to transfect cells?

Answer 40

Electroporation calcium phosphate precipitation lipid mediated lipofection retroviral infection microinjection DEAE- dextran

Question 41

how does electroporation work?

Answer 41

membrane becomes highly permeable and more positivley charged, negative DNA crosses more easily into cell

Question 42

pros/cons of electroporation?

Answer 42

nonchemical method (doesn't alter biochemical structure) easy effeicient cell mortality (if suboptimal conditions)

Question 43

how were cells split to ensure 70% confluecny at time of transfection via electroporation?

Answer 43

split 1:3

Question 44

what are the components of the lysis buffer for the cell lysates?

Answer 44

Tris HCL- buffer Sucrose- osmotic pressure EDTA/EGTA- protease inhibitors, chelate Mg+, Ca+ Sodium flourode/Sodium pyrophosphate- protease inhibitors Triton X-100- detergent, permeabilises cell membrane

Question 45

why were the cell lysates kept on ice?

Answer 45

slow down denaturation of protease

Question 46

why is B-Mercaptoethanol added to cell lysates?

Answer 46

reduces disulphate bridges- proteins can separate on W. Blot

Question 47

What are the three elements of a Western Blot?

Answer 47

Separation by size trasnfer to solid support marking of target protein

Question 48

what are PAGE gels and why are they a hazard?

Answer 48

polyacrylamide gel electrophoresis can contain unploymerised acrylamide which is a neurotoxin- need ot be disposed of in cytotoxic bin

Question 49

what was the gradient of PAGE gel used?

Answer 49

4-12% lower % of acyrlamide at top of gel and high % at bottom

Question 50

the proteins have what charge when runnign W. Blot?

Answer 50

negatively charged run from -ve electrode to +ve electrode

Question 51

how are the proteins separated during W. Blot?

Answer 51

separated by weight- kDa

Question 52

how long were the W. Blot gels run for?

Answer 52

60mins | (keep on ice to avoid overheating)

Question 53

what is used for immunoblotting of the proteins follwoing western blot?

Answer 53

nitrocellulose membranes electrophoresis

Question 54

during electrophoresis proteins migrate from the gel onto the nitrocellulose membrane. why do they bind to the membrane?

Answer 54

hydrophobic interactions

Question 55

why is marvel used?

Answer 55

masks isotopes so prevents binding of antibody to preoteins non-specifically

Question 56

what is the primary antibody?

Answer 56

specific to GFP or GAPDH

Question 57

what is the secondary antibody?

Answer 57

bonds to both primary antibodies conjugated to HRP (horse radish peroxidase)

Question 58

is GFP baturally found in COS7 cells?

Answer 58

No- ill only be expressed in those trasnfected with plasmid DNA

Question 59

what was our housekeeping protein?

Answer 59

GAPDH, naturally found in all cells

Question 60

What is ECL?

Answer 60

Electrochemiluminescence

Question 61

what are the two solutions used to create ECL?

Answer 61

Hydrogen peroxidase luminol

Question 62

how does ECL emit a light signal?

Answer 62

luminal is oxidised when contact with HRP and so becomes excited- emits light when it falls back down to baselinedetected by x-ray film

Question 63

how long does ECL last?

Answer 63

1 hr

Question 64

what is PCR?

Answer 64

amplification of small segments of DNA

Question 65

what is the enzyme used in PCR?

Answer 65

Taq Polymerase synthesizes two new strands of DNA using originals as templates

Question 66

what are the steps involved in PCR?

Answer 66

denaturation- 93C annealing- 61C elongation- 72C

Question 67

what is sybersafe?

Answer 67

DNA gel stain that flouresces under UV light

Question 68

what type of PCR was done to determien is kRAS mutation was present?

Answer 68

genotyping PCR | (wild type and het)