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BMSc > Labs > Flashcards

Labs Flashcards

1
Q

What are the differnet micropipette volumes?

A

P10

P20

P200

P1000

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2
Q

how do you balance a centrifuge?

A

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

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3
Q

what is molarity?

A

measure of concentration of solute in solution

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4
Q

what are moles?

A

molecular weight of a substance expressed in grams

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5
Q

molarity = ?

A

moles per litre

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6
Q

distinguish between moles and molarity

A

moles- quantity of material

molarity- concentration of material in solution

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7
Q

what is avagadros?

A

one mole of substance

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8
Q

what calculation is used for stock solutions?

A

C1V1= C2V2

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9
Q

what is a lowry protein assay?

A

determine protein concentration in unknown sample

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10
Q

what reagent is used for colour chnage in lowry?

what is the colour change?

A

folins reagent

yellow/brown to blue

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11
Q

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

A

spectrophotometer

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12
Q

describe in vitro/ in vivo

A

in vitro- in glass

in vivo- in life

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13
Q

which cell type was used in tissue culture?

A

COS7 cells

Fibroblastic cells from monkey kidney

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14
Q

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

A

sub culture or passage

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15
Q

what is passaging?

A

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

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16
Q

what are the growth phases of cell during tissue culture?

A

lag phase

log phase- proliferation of cells

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17
Q

how often were the cells cultured?

A

twice a week

Mon/Fri

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18
Q

what is PBS?

A

Phosphate Buffered Saline

used to wash the cells

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19
Q

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

A

Trypsin EDTA

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20
Q

What growth medium was used in tissue culture?

A

DMEM media

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21
Q

what were the components of the DMEM?

A

10% FBS (extra nutrients)

glutamine- essential amino acid

sodium pyruvate- carbon source

1% penstrep- prevent infection

phenyl red dye- pH indicator

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22
Q

what is confluency?

what % is best when passaging in culture?

A

coverage of dish by cells

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

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23
Q

What in the DMEM media inhibits trypsin activty?

A

calcium

thsi is why cells are washed with PBS

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24
Q

describe a 1:5 split

A

2ml cell suspension into new dish + 8ml DMEM

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25
Q

What is GFP?

A

Green Flourescent Protein

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26
Q

What bacteria were used for culture?

A

E.coli

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27
Q

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

A

LB broth containing candymiycin

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28
Q

what is a plasmid?

A

double stranded, circular extra chomosomal DNA of bacterium

29
Q

what method was used to isolate the plasmid DNA?

A

alkaline lysis

30
Q

describe isolation of plasmid

A

cell growth and harvest

re-suspension (TRIS, EDTA, Glucose)

lysis (SDS, NaOH)

neutralisation (potassium acetate)

cleanign and concentration (ethanol)

31
Q

what are the components of alkaline lysis buffer?

A

SDS detergent- cleaves phospholipid bilayer of membrane

Alkali- NaOH, denatures structural proteins of cell membrane and breaks hydrogen bonds

32
Q

what was in the suspension solution for plasmid DNA?

A

TRIS- buffer

Glucose- osmotic pressure

EDTA- chelates divalent cations Mg+, Ca+

33
Q

what does the potassium acetate do during alkaline lysis?

A

neutralises solution allowing hydrogen bonds between bases to re-establish

34
Q

why is addition of potassium acetate important?

A

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)

35
Q

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

A

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

36
Q

what is used to seperate the DNA?

A

isopropanol

37
Q

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

A

ethanol precipitation

38
Q

what is transfection?

A

introdunction of foreign DNA into nucleus of eukaryotic cells

39
Q

what are the two types of transfectant?

A

stable- cells have intregratde foreign DNA

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

40
Q

what methods can be used to transfect cells?

A

Electroporation

calcium phosphate precipitation

lipid mediated lipofection

retroviral infection

microinjection

DEAE- dextran

41
Q

how does electroporation work?

A

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

42
Q

pros/cons of electroporation?

A

nonchemical method (doesn’t alter biochemical structure)

easy

effeicient

cell mortality (if suboptimal conditions)

43
Q

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

A

split 1:3

44
Q

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

A

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

45
Q

why were the cell lysates kept on ice?

A

slow down denaturation of protease

46
Q

why is B-Mercaptoethanol added to cell lysates?

A

reduces disulphate bridges- proteins can separate on W. Blot

47
Q

What are the three elements of a Western Blot?

A

Separation by size

trasnfer to solid support

marking of target protein

48
Q

what are PAGE gels and why are they a hazard?

A

polyacrylamide gel electrophoresis

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

49
Q

what was the gradient of PAGE gel used?

A

4-12%

lower % of acyrlamide at top of gel and high % at bottom

50
Q

the proteins have what charge when runnign W. Blot?

A

negatively charged

run from -ve electrode to +ve electrode

51
Q

how are the proteins separated during W. Blot?

A

separated by weight- kDa

52
Q

how long were the W. Blot gels run for?

A

60mins

(keep on ice to avoid overheating)

53
Q

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

A

nitrocellulose membranes

electrophoresis

54
Q

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

A

hydrophobic interactions

55
Q

why is marvel used?

A

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

56
Q

what is the primary antibody?

A

specific to GFP or GAPDH

57
Q

what is the secondary antibody?

A

bonds to both primary antibodies

conjugated to HRP (horse radish peroxidase)

58
Q

is GFP baturally found in COS7 cells?

A

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

59
Q

what was our housekeeping protein?

A

GAPDH, naturally found in all cells

60
Q

What is ECL?

A

Electrochemiluminescence

61
Q

what are the two solutions used to create ECL?

A

Hydrogen peroxidase

luminol

62
Q

how does ECL emit a light signal?

A

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

63
Q

how long does ECL last?

A

1 hr

64
Q

what is PCR?

A

amplification of small segments of DNA

65
Q

what is the enzyme used in PCR?

A

Taq Polymerase

synthesizes two new strands of DNA using originals as templates

66
Q

what are the steps involved in PCR?

A

denaturation- 93C

annealing- 61C

elongation- 72C

67
Q

what is sybersafe?

A

DNA gel stain that flouresces under UV light

68
Q

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

A

genotyping PCR

(wild type and het)

BMSc (2 decks)

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