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RA Interview > Techniques > Flashcards

Techniques Flashcards

1
Q

What is confocal microscopy?

A
  • rejects out of focus light from detector (as opposed to FM) so does not blur images
  • allowing high res imaging of eg. thick tissues
  • by using spot that is moved over sample to collect data point by point
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2
Q

What is resolution (microscopy)?

A

Min distance at which 2 points can be distinguished in defined space

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3
Q

What is exposure time (microscopy)?

A

Length of time light collected to produce image

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4
Q

What is gain (microscopy)?

A

Digital amp of data at particular exposure to boost weak signals

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5
Q

What is refraction (microscopy)?

A

The change in direction of light when passing from one medium to another

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6
Q

What are parafocal objectives (microscopy)?

A

Means if focus image correctly, when move to higher magnification objective should remain close to focal plane

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7
Q

What causes saturation (microscopy)?

A

If exposure times too long, areas can become saturates where the sensor chip cannot absorb anymore light

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8
Q

What is the biggest risk to cell culture?

A

Contamination of cell stocks

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9
Q

What are the diff types of cell culture contamination?

A
  • chemical
  • human
  • viral
  • bacterial
  • intracellular bacteria
  • fungal
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10
Q

What causes chemical contamination in cell culture and how can it be identified?

A
  • incorrectly adding reagent
  • calculation errors so wrong amount
  • hard to idenitfy until signs of cellular stress visible
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11
Q

What causes human cell contamination in cell culture and how can it be identified?

A
  • mistakes when passaging multiple cell lines and mixing up
  • heterogenous pop hard to identify unless cell lines have distinct morphologies or results suddenly change
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12
Q

What causes viral contamination in cell culture and how can it be identified?

A
  • v hard to identify as not visible and require specific testing
  • often little impact on cells but severe infection can include changes in cell morphology/behaviour
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13
Q

What causes bacterial contamination in cell culture and how can it be identified?

A
  • most common and easily identifiable
  • inspect flasks to see if cloudy
  • visualisable earlier on under microscopy, usually as small dark cylindrical cells
  • causes physiological stress to cells which can affect results
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14
Q

What causes intracellular bacterial contamination in cell culture and how can it be identified?

A
  • eg. mycoplasma lack cell wall so resistant to antibiotics that target this
  • proliferate in euk cells and affect results
  • too small to observe, identify by diagnostic PCR
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15
Q

What causes fungal contamination in cell culture and how can it be identified?

A
  • yeast small uniform and appear bright under phase microscopy
  • other fungi form individual large growths with hyphae
  • microscopic analysis to monitor
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16
Q

What does aseptic technique involve?

A
  • laminar flow hood, pull outside air down away from working surface, HEPA filter to prevent contaminants
  • organised hood
  • extraction fan
  • spray all surfaces w/ ethanol, plus anything that enters hood including gloves
17
Q

What components can be inc in cell culture media?

A
  • base solution of electrolytes, AAs, vitamins, sugars
  • phenol red: assess acidity as indicator of metabolic waste produced
  • FBS: provides macromolecular proteins, hormones, growth factors and stimulates cell growth and prolif
18
Q

What is confluency and why must it be monitored?

A
  • visually inspect flasks regularly to assess confluency
  • when optimum balance between health and number of cells in culture achieved
  • after this cell numbers increasing will negatively affect cell health
19
Q

What is passaging?

A
  • taking small sample of cells from confluent culture and setting up new culture
  • excess of space and nutrients will initiate proliferation cycle again
20
Q

What happens if don’t passage cells?

A
  • excess of cells will exhaust nutrients in media and cause physiological stress
  • drives culture adaption, affect results, cell death and loss of cell line
21
Q

Basic passaging protocol (adherent)?

A
  • aspirate off culture media
  • wash cells in PBS
  • add trypsin and incubate to detach cells
  • quench trypsin with media
  • transfer to falcon and centrifuge
  • aspirate off culture media and resuspend in media
  • count cells, calculate conc, and total cell no.
  • seed a vol of cell suspension into new flask and add fresh media
  • incubate
22
Q

How do you calculate culture viability?

A
  • add Tryphan blue, dead cells have increased membrane permeability
  • viability (%) = total live/total cells x100
23
Q

What are the 3 steps of PCR?

A

1) Denaturation (>90) - dsDNA templates heated to separate strands
2) Annealing (55-70)- primers bind to flanking regions of target DNA
3) extension (70) - DNA pol extends 3’ end of each primer along template strands
Repeated 25-35 times to exponentially produce copies of target

24
Q

Why is Taq pol used?

A

Thermostable over continuous cycling (up to around 90)

25
Q

Disadvantages of Taq polymerase?

A
  • relatively unstable over 90 during denaturation, esp problem for templates w/ high GC content and/or strong secondary structures
  • lacks proofreading activity, can misincorporate nucleotides
  • cannot amp fragments >5kb
26
Q

What are the key components of a PCR reaction?

A
  • DNA pol
  • primers
  • DNA to be amp
  • dNTPs
  • water, buffers
  • Mg ions as cofactor, enhances pol activity and increases yield
27
Q

General rules for designing PCR primers?

A
  • 15-30nt
  • Tm 55-70
  • 40-60% GC
  • C/G at 3’ end (no more than 3)
  • no complementarities to cause secondary structures
  • no direct repeats
28
Q

What could cause no/low amp in PCR

A
  • DNA templates: poor integrity, low purity, insufficient quantity DNA, complex target (eg. high GC), long target
  • primers: problematic design and specificity, old primers, insufficient quantity
  • other reaction components: inapprop pol (use hot start), insufficient pol, insufficient MG ions, excess additives, dUTP in reaction mix, nonhomogenous reagents
  • thermal cycling conditions: suboptimal denaturation times (short not enough to separate, but long may reduce enzyme activity), suboptimal annealing temp, suboptimal extension time, suboptimal no. cycles
29
Q

What could cause nonspecific amplification in PCR?

A
  • DNA templates: excess DNA input, poor integrity, complex seqs, long targets
  • primers: problematic design, high quantity
  • other reaction components: excess pol, inapprop pol, excess Mg ions
  • thermal cycling conditions: insufficient denaturation, incorrect annealing temp for pol, low annealing temp, high extension temp, insufficient extension temp, too many cycles
30
Q

What can cause sequence errors in PCR?

A
  • low fidelity DNA pol
  • excess Mg ions
  • unbalanced dNTPs
  • high no. cycles
  • UV damaged DNA
  • sequencing error
31
Q

Basic workflow of mol cloning?

A
  • isolate target DNA fragments
  • ligate inserts into approp cloning vector, creating recombinant mol
  • transform recombinant plasmid into bacteria/host
  • screening of hosts containing recombinant plasmid
32
Q

What features should mutagenesis primers have?

A
  • 10-15bp of flanking region each side of the mutation
  • total primer size 20-30bp
  • avoid repeats/palindromic seqs
  • forward and reverse primers complimentary
  • similar melting temps
  • min 40% GC content
  • GC clamp in last 5nts
  • correct mismatch and central in primer
33
Q

How can newly synthesised plasmid seqs be distinguished in SDM?

A
  • do not contain methylated DNA as prod in vitro
  • methylation can only occur within a bacterial cell
  • adding RE DpnI to completed PCR will selectively degrade methylated template
34
Q

How do western blots work?

A
  • separate mix of proteins based of mol weight, so also by type, through gel electrophoresis
  • results transferred to a membrane prod band for each protein, then incubated w/ labelled Abs specific to protein of interest
  • unbound Ab washed off, so only band for protein of interest present
  • size of band indicates amount present
35
Q

How is a western blot set up?

A
  • from -ve to +ve: sponge, filter papers, gel, blotting membrane, filer papers, sponge
  • wet transfer from -ve plate
36
Q

How does immunoprecipitation work?

A
  • affinity purification of antigens w/ specific antibodies immobilised to magnetic particles or agarose resin
  • used to isolate proteins for subsequent detection by WB/other assays

RA Interview (1 decks)

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