Techniques Flashcards
What is confocal microscopy?
- 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
What is resolution (microscopy)?
Min distance at which 2 points can be distinguished in defined space
What is exposure time (microscopy)?
Length of time light collected to produce image
What is gain (microscopy)?
Digital amp of data at particular exposure to boost weak signals
What is refraction (microscopy)?
The change in direction of light when passing from one medium to another
What are parafocal objectives (microscopy)?
Means if focus image correctly, when move to higher magnification objective should remain close to focal plane
What causes saturation (microscopy)?
If exposure times too long, areas can become saturates where the sensor chip cannot absorb anymore light
What is the biggest risk to cell culture?
Contamination of cell stocks
What are the diff types of cell culture contamination?
- chemical
- human
- viral
- bacterial
- intracellular bacteria
- fungal
What causes chemical contamination in cell culture and how can it be identified?
- incorrectly adding reagent
- calculation errors so wrong amount
- hard to idenitfy until signs of cellular stress visible
What causes human cell contamination in cell culture and how can it be identified?
- mistakes when passaging multiple cell lines and mixing up
- heterogenous pop hard to identify unless cell lines have distinct morphologies or results suddenly change
What causes viral contamination in cell culture and how can it be identified?
- 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
What causes bacterial contamination in cell culture and how can it be identified?
- 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
What causes intracellular bacterial contamination in cell culture and how can it be identified?
- 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
What causes fungal contamination in cell culture and how can it be identified?
- yeast small uniform and appear bright under phase microscopy
- other fungi form individual large growths with hyphae
- microscopic analysis to monitor
What does aseptic technique involve?
- 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
What components can be inc in cell culture media?
- 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
What is confluency and why must it be monitored?
- 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
What is passaging?
- taking small sample of cells from confluent culture and setting up new culture
- excess of space and nutrients will initiate proliferation cycle again
What happens if don’t passage cells?
- excess of cells will exhaust nutrients in media and cause physiological stress
- drives culture adaption, affect results, cell death and loss of cell line
Basic passaging protocol (adherent)?
- 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
How do you calculate culture viability?
- add Tryphan blue, dead cells have increased membrane permeability
- viability (%) = total live/total cells x100
What are the 3 steps of PCR?
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
Why is Taq pol used?
Thermostable over continuous cycling (up to around 90)
Disadvantages of Taq polymerase?
- 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
What are the key components of a PCR reaction?
- DNA pol
- primers
- DNA to be amp
- dNTPs
- water, buffers
- Mg ions as cofactor, enhances pol activity and increases yield
General rules for designing PCR primers?
- 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
What could cause no/low amp in PCR
- 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
What could cause nonspecific amplification in PCR?
- 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
What can cause sequence errors in PCR?
- low fidelity DNA pol
- excess Mg ions
- unbalanced dNTPs
- high no. cycles
- UV damaged DNA
- sequencing error
Basic workflow of mol cloning?
- 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
What features should mutagenesis primers have?
- 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
How can newly synthesised plasmid seqs be distinguished in SDM?
- 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
How do western blots work?
- 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
How is a western blot set up?
- from -ve to +ve: sponge, filter papers, gel, blotting membrane, filer papers, sponge
- wet transfer from -ve plate
How does immunoprecipitation work?
- 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
