iron chelators - trap iron to prevent enzymatic reactions - prevents DNA damage - destabilises membrane

Lab skills Flashcards by Sana Nizam

What affects pipetting?

temperature
viscosity
tips fall down

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Reagent prep

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Lowry standard curve

Linear relationship

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Why was the protein diluted in Lowry?

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

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1:100 dilution

Outwith spectrophotometer limits

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Plasmids have a ____________ resistant gene in their DNA

kanamycin

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Plasmid structure

circular dsDNA

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Plasmid normal function

Bacterial resistance

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Glucose maintains _______.
Tris HCl buffers maintain _______.

osmotic pressure
pH (8)

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EDTA

iron chelators - trap iron to prevent enzymatic reactions

prevents DNA damage
destabilises membrane

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PBS

removes antitrypsin in EDTA

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NaOH and SDS in plasmid extraction

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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SDS

denaturate proteins in cells

destabilises membrane

denatures enzymes

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Neutralisation

Brings pH closer to 7

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What does the supernatant contain?

sds
ssdna
genomic dna

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Isopropanol before spinning

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

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70% ethanol before spinning

Precipitate DNA and salt

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Pellet

Concentrates plasmid for further experiments

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cDNA

complementary DNA reverse transcribed from RNA (no introns)

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Purpose of passaging

make space
add nutrients
remove toxins

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What does DMEM contain?

vitamins
GF
glucose
minerals
amino acids
salts

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Limitation of pen strep

Doesn’t prevent fungal or yeast contamination

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Uses of cell counting

transfection

drug impact

comparison of confluence between experiment repeats

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Cell counting split

1:3 instead of 1:5

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What cell confluence is best for DNA uptake?

70% - actively dividing

How does electroporation work?

Membrane pores open Cells become positively charged, allowing negative DNA inside Membrane pores close DNA transcribed and protein expressed

PCR

Amplifies small segments of DNA

Taq polymerase

Bacteria found in water

What temperature does Taq polymerase work at?

32 degrees optimum can work in high temperatures Denaturation at 94*C

dNTPs

dATP, dGTP, dCTP, dTTP

Primers

18-24 nucleotides that specifically amplify DNA

PCR number of cycles

30-40

PCR steps

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

Cybersafe

fluorescent under UV when it binds to DNA

% of _________ affects runs

agarose

What is the ladder used for

Making sure the DNA is the right size

Lysis buffer

Solution used to break open cells to allow protein collection

B mercaptoethanol function in lysis buffer

break all the disulfide bonds and denature the protein of interest

What does the lysis buffer contain?

b mercaptoethanol protease inhibitor

Is the western blot qualitative or quantitative?

Semi quantitative

Which side is the membrane on?

Anode

What does ponceau staining show?

air bubbles, protein transfer

Why is milk added to the membrane?

To prevent protease from binding to the charged membrane

Primary antibodies

Rabbit anti GFP and rabbit anti GAPDH

What is the purpose of a loading control?

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

Why is GAPDH used instead of other proteins?

stable expression in cells (other proteins vary according to cells and conditions)

Secondary antibody

Rabbit IgG conjugated to HRP enzyme

H2O2

oxidizing substrate of horseradish peroxidase

GFP band position

26 kda

Mock

water instead of transfected with cells

What are plasmids used for

Clone genes and make large quantities

Plasmid size

1-1000 k bp

Steps of extracting plasmids

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

How is the bacteria lysed?

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

Detergent effect on membrane

Cleaves phospholipid bilayer

Alkali effect on membrane

Denatures proteins

How is cellular debris removed?

Precipitatation Centrifusion Removal of supernatent

Plasmid extraction: solution 1

Glucose TRIS HCl EDTA

Plasmid extraction: solution 2

SDS and NaOH

Plasmid extraction: solution 3 (neutralisation)

Potassium acetate Glacial acetic acid

PCR uses

Fingerprinting AIDS detection Genetic diagnosis

How long does a thermocycler take

Few hours

Each PCR tube

1 ul forward primer 1 ul reverse primer 22 ul master mix 1 ul DNA or water

Master mix

MgCl2 Taq dNTPs

Trypsin blue dye permeates _____ cells

dead

PAGE gel danger

Unpolymerised acrylamide is a neurotoxin

Copper ions donated from CuSO4

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

NaOH

alkaline medium

K/Na tartrate

chelating agent that stabilises the copper

Reduced copper

Reduces Folin’s reagent

Folin’s reagent

Cu1+ to Cu2+

Potassium acetate

Precipitation of a SDS-protein complex Neutralisation, allowing renaturation

PCR denaturation temperature and time

95 °C for 1 min

PCR annealing temperature and time

59 °C for 30 s

PCR extension temperature and time

72 °C for 1 min

Cell culture growth phases

lag phase to log phase, where the cells proliferate exponentially

How long are transient transfectants expressed for?

24-96 hours

4-12% gradient gel

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

Done!

Lab skills Flashcards

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