Lab skills Flashcards
What affects pipetting?
temperature
viscosity
tips fall down
Reagent prep
CV
Lowry standard curve
Linear relationship
Why was the protein diluted in Lowry?
To ensure it wasn’t on the plateau (beyond the maximum detectable concentration)
1:100 dilution
Outwith spectrophotometer limits
Plasmids have a ____________ resistant gene in their DNA
kanamycin
Plasmid structure
circular dsDNA
Plasmid normal function
Bacterial resistance
Glucose maintains _______.
Tris HCl buffers maintain _______.
osmotic pressure
pH (8)
EDTA
iron chelators - trap iron to prevent enzymatic reactions
- prevents DNA damage
- destabilises membrane
PBS
removes antitrypsin in EDTA
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
SDS
denaturate proteins in cells
destabilises membrane
denatures enzymes
Neutralisation
Brings pH closer to 7
What does the supernatant contain?
sds
ssdna
genomic dna
Isopropanol before spinning
Lessens the risk that solutes like sucrose or NaCl will be coprecipitated with the DNA
70% ethanol before spinning
Precipitate DNA and salt
Pellet
Concentrates plasmid for further experiments
cDNA
complementary DNA reverse transcribed from RNA (no introns)
Purpose of passaging
make space
add nutrients
remove toxins
What does DMEM contain?
vitamins
GF
glucose
minerals
amino acids
salts
Limitation of pen strep
Doesn’t prevent fungal or yeast contamination
Uses of cell counting
transfection
drug impact
comparison of confluence between experiment repeats
Cell counting split
1:3 instead of 1:5
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
- Denaturation - unwinding
- Annealing - primers attach to DNA
- 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
- Growth of bacterial cell culture
- Harvesting and lysis
- 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!
