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Bmsc > Lab skills > Flashcards

Lab skills Flashcards

1
Q

What affects pipetting?

A

temperature
viscosity
tips fall down

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2
Q

Reagent prep

A

CV

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3
Q

Lowry standard curve

A

Linear relationship

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4
Q

Why was the protein diluted in Lowry?

A

To ensure it wasn’t on the plateau (beyond the maximum detectable concentration)

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5
Q

1:100 dilution

A

Outwith spectrophotometer limits

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6
Q

Plasmids have a ____________ resistant gene in their DNA

A

kanamycin

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7
Q

Plasmid structure

A

circular dsDNA

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7
Q

Plasmid normal function

A

Bacterial resistance

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8
Q

Glucose maintains _______.
Tris HCl buffers maintain _______.

A

osmotic pressure
pH (8)

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9
Q

EDTA

A

iron chelators - trap iron to prevent enzymatic reactions

  • prevents DNA damage
  • destabilises membrane
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10
Q

PBS

A

removes antitrypsin in EDTA

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11
Q

NaOH and SDS in plasmid extraction

A

pH of 12-12.5 irreversibly denatures genomic linear DNA but the plasmid is only slightly denatured

Converts dsDNA to ssDNA

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12
Q

SDS

A

denaturate proteins in cells

destabilises membrane

denatures enzymes

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13
Q

Neutralisation

A

Brings pH closer to 7

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13
Q

What does the supernatant contain?

A

sds
ssdna
genomic dna

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14
Q

Isopropanol before spinning

A

Lessens the risk that solutes like sucrose or NaCl will be coprecipitated with the DNA

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15
Q

70% ethanol before spinning

A

Precipitate DNA and salt

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16
Q

Pellet

A

Concentrates plasmid for further experiments

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17
Q

cDNA

A

complementary DNA reverse transcribed from RNA (no introns)

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18
Q

Purpose of passaging

A

make space
add nutrients
remove toxins

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19
Q

What does DMEM contain?

A

vitamins
GF
glucose
minerals
amino acids
salts

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20
Q

Limitation of pen strep

A

Doesn’t prevent fungal or yeast contamination

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21
Q

Uses of cell counting

A

transfection

drug impact

comparison of confluence between experiment repeats

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22
Q

Cell counting split

A

1:3 instead of 1:5

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23
Q

What cell confluence is best for DNA uptake?

A

70% - actively dividing

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24
Q

How does electroporation work?

A

Membrane pores open

Cells become positively charged, allowing negative DNA inside

Membrane pores close

DNA transcribed and protein expressed

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25
Q

PCR

A

Amplifies small segments of DNA

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26
Q

Taq polymerase

A

Bacteria found in water

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27
Q

What temperature does Taq polymerase work at?

A

32 degrees optimum

can work in high temperatures
Denaturation at 94*C

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28
Q

dNTPs

A

dATP, dGTP, dCTP, dTTP

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29
Q

Primers

A

18-24 nucleotides that specifically amplify DNA

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30
Q

PCR number of cycles

A

30-40

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31
Q

PCR steps

A
  1. Denaturation - unwinding
  2. Annealing - primers attach to DNA
  3. Extension - by DNTPs
32
Q

Cybersafe

A

fluorescent under UV when it binds to DNA

32
Q

% of _________ affects runs

A

agarose

33
Q

What is the ladder used for

A

Making sure the DNA is the right size

34
Q

Lysis buffer

A

Solution used to break open cells to allow protein collection

35
Q

B mercaptoethanol function in lysis buffer

A

break all the disulfide bonds and denature the protein of interest

36
Q

What does the lysis buffer contain?

A

b mercaptoethanol

protease inhibitor

37
Q

Is the western blot qualitative or quantitative?

A

Semi quantitative

38
Q

Which side is the membrane on?

A

Anode

39
Q

What does ponceau staining show?

A

air bubbles, protein transfer

40
Q

Why is milk added to the membrane?

A

To prevent protease from binding to the charged membrane

41
Q

Primary antibodies

A

Rabbit anti GFP and rabbit anti GAPDH

42
Q

What is the purpose of a loading control?

A

To check that the same amount of protein has been added to each well

43
Q

Why is GAPDH used instead of other proteins?

A

stable expression in cells

(other proteins vary according to cells and conditions)

44
Q

Secondary antibody

A

Rabbit IgG conjugated to HRP enzyme

45
Q

H2O2

A

oxidizing substrate of horseradish peroxidase

46
Q

GFP band position

A

26 kda

47
Q

Mock

A

water instead of transfected with cells

48
Q

What are plasmids used for

A

Clone genes and make large quantities

49
Q

Plasmid size

A

1-1000 k bp

50
Q

Steps of extracting plasmids

A
  1. Growth of bacterial cell culture
  2. Harvesting and lysis
  3. Purification
51
Q

How is the bacteria lysed?

A

With an alkaline lysis buffer consisting of detergent sodium dodecyl sulphate and a strong base sodium hydroxide

52
Q

Detergent effect on membrane

A

Cleaves phospholipid bilayer

53
Q

Alkali effect on membrane

A

Denatures proteins

54
Q

How is cellular debris removed?

A

Precipitatation
Centrifusion
Removal of supernatent

55
Q

Plasmid extraction: solution 1

A

Glucose
TRIS HCl
EDTA

56
Q

Plasmid extraction: solution 2

A

SDS and NaOH

57
Q

Plasmid extraction: solution 3 (neutralisation)

A

Potassium acetate
Glacial acetic acid

58
Q

PCR uses

A

Fingerprinting
AIDS detection
Genetic diagnosis

59
Q

How long does a thermocycler take

A

Few hours

60
Q

Each PCR tube

A

1 ul forward primer

1 ul reverse primer

22 ul master mix

1 ul DNA or water

61
Q

Master mix

A

MgCl2
Taq
dNTPs

62
Q

Trypsin blue dye permeates _____ cells

A

dead

63
Q

PAGE gel danger

A

Unpolymerised acrylamide is a neurotoxin

64
Q

Copper ions donated from CuSO4

A

Interact with amide and peptide bonds in the protein to form a light-absorbing molecule

65
Q
A
66
Q

NaOH

A

alkaline medium

66
Q

K/Na tartrate

A

chelating agent that stabilises the copper

67
Q

Reduced copper

A

Reduces Folin’s reagent

68
Q

Folin’s reagent

A

Cu1+ to Cu2+

69
Q

Potassium acetate

A

Precipitation of a SDS-protein complex

Neutralisation, allowing renaturation

70
Q

PCR denaturation temperature and time

A

95 °C for 1 min

71
Q

PCR annealing temperature and time

A

59 °C for 30 s

72
Q

PCR extension temperature and time

A

72 °C for 1 min

73
Q

Cell culture growth phases

A

lag phase to log phase, where the cells proliferate exponentially

74
Q

How long are transient transfectants expressed for?

A

24-96 hours

75
Q

4-12% gradient gel

A

lower percentage of acrylamide at the top and a high percentage at the bottom, enabling a broad range of protein sizes to be separated

76
Q

Done!

A

Bmsc (17 decks)

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