Practicals Flashcards
State important design features of a primer
3’ CG clamp
20bp minimum length
Equal distribution of A,T,C,G
No palindromes
No homopolymeric sequences
No self complementary regions
How is the melting temperature (Tm) calculated?
Wilson’s Rule
2(A+T)+ 4(C+G)
How is the annealing temperature calculated?
-2 degrees from melting temperature
What is the reason for 94, 48 and 72 degrees being used in PCR?
94 denaturation of DNA strands
48 annealing temperature
72 primer extension, replication temp
How does ethidium bromide allow you to visualize a gel electrophoresis?
It intercalates between DNA bases
When bound
Becomes fluorescent under UV
How to calculate the amount of product produced from a PCR?
1) Calculate efficiency
2) Multiply by 2^ (cycles)
Suggest one simple strategy by which mis-priming can be alleviated
Hot-start
Increase annealing temperature
(If Mg2+ used, decrease conc of Mg2+, because it is present to stabilize DNA)
What is the definition of spatial resolving power?
Ability to distinguish between two closely located objects as two separate entities
How can RNA and DNA be stained?
RNA- Pyronin (pink)
DNA- DAPI (blue)
What is a somite?
Cells that give rise to ribs and spine
Drosophila (synchronous segmentation)
Chick (sequential segmentation)
How do you determine the single trait ratio?
+/-
How would you test if a mutation is in a single gene?
Backcross mutant with WT
expect a 1:1
How would you test whether a mutation is on the same gene or not?
Complementation analysis
If gene on the same, crossing would lead to death most likely
However if not, then offspring will be OK, because the genes complement each other
What is epistasis and how does it differ from dominance?
epistasis- masking of one gene by another
Dominance- masking of an allele by another, but on the same gene
What aspects of the Aspergillus nidulans (fungus) life cycle make it a useful model organism for the genetic analysis of a metabolic pathway?
Form heterokaryons
Haploid so don’t show dominance
Produces many progeny
Easy to grow
How would you test whether a mutation in Saccharomyces cerevisiae is derived from a mutation in the nuclear gene or a mitochondrial gene?
Cross with WT
1:1 is nuclear
Uneven distribution if mitochondrial
What is the Shine- Dalgarno sequence?
Ribosome binding sequence
Located 8bp before AUG start codon
Helps recruit ribosome
How to tell whether the gene will be efficiently transcribed?
If the sequence doesn’t differ much from the consensus sequence
Why is ice cold Calcium chloride used in the transformation of bacteria?
Speed up the process of plasmid transformation
cold shock required
Overall increasing efficiency
What control would need to be implemented to show that the results are due to the plasmids and not by contamination?
Carry out same experiment in another tube
However don’t add plasmids to that tube
What is the purpose of rapid heating and cooling of the lysate, in plasmid extraction?
Rapid heating causes H bonds and membrane to break
Rapid cooling H bonds reform, however chromosomal being so large and long won’t reform properly, so won’t be in solution
Can be centrifuged out
In the supernatant remains the plasmid DNA, which successfully rebonded
Why is there a high concentration of mannitol in the buffer?
Acts as an isotonic buffer
Stop osmotic lysis
What does pKa mean?
How acidic an atom of H is
-logKa
Low pKa= strong acid
Describe in detail the method of staining pancreatic acinar cells
1) Fix cells to the slide, dehydrate set in paraffin wax
2) remove wax by adding Histo-clear
3) Wash in 100% then 70% alcohol
4) Rinse
5)Methyl green/ pyronin stain (30min)
6) Blot dry
7) Dry for an hour
8) Add mounting medium and cover slip
9) Place on hot plate to seal
How would you set up a light microscope?
1) Focus slide, adjust the brightness
2) Set condenser iris into the middle
3) Close field iris until a hexagon appears
4) Centre the hexagon using condenser screws
5) Focus hexagon by using condenser focus
6) Open the field iris until the hexagon begins to disappear
7) Remove an eyepiece
8) Close the condenser iris until circle is 2/3 of the hexagon
8) Replace the eye piece
How are bird RBC different to human RBC?
Bird RBC have a nucleus
How would you estimate the size of a cell?
use graticule
Know the dimensions that correlate to the magnification
Describe the approach you would use to extract soluble protein from a plant
Take a disk sample ,using cork borer
Homogenise in 50ul phosphate buffer
Store on ice
ultracentrifuge for 2 min
discard pellet
Store on ice
What technical factors might be responsible for the low value reading on absorbance?
low leaf harvest
Inaccurate pipetting
Misplacing the cuvette
Not adding Bradfords reagent
Poor grinding
What is the principle on which the Bradford’s assay is based?
Colometric assay
Coomassie dye bonding with Arg and hydrophobic residues
Expression based on presence of protein
Calculate the rise per amino acid
3.5 amino acids per turn
Count the number of turns
Multiply turns by 3.5
Divide whole length by the number
An area of protein has no secondary structure suggest why
Gives protein flexibility
If close to ligand binding site
Allows for conformational change
Suggest why hydrophobic residues are important in the function of a protein as a receptor
Energetically unfavourable to expose to outside
Encourages receptors to bind to proteins/ chemicals
Explain how you would distinguish between a male and a female drosophila
Male- dark abdomen
Female - lighter abdomen, usually larger also
Why is it important to use a virgin female in the cross?
Females can store sperm
So may be unsure who the farther of the progeny is
As she may have stored sperm from brother
Briefly explain how you would test antibiotic resistance in a bacteria
Spread antibiotic on agar place
Streak/patch bacteria onto plate
Incubate for a week
Analyse growth
If growth has occurred then resistant
Have a positive control, plate with no antibiotic, as a control phenotype for growth
What is melting temperature?
When 50% of primers are bound and 50% are unbound
What is tubulin’s role in the cell?
Polymerises to microtubules
Polarised
Control movement of proteins
Controls location and alignment of chromosomes in the middle during metaphase
Controls their movement to the poles in anaphase
How would you prepare cells for immunofluorescent microscopy?
Cells fixed to the slide
Antibody complementary to the organelle added
Secondary antibody attached to the dye, complementary to the first antibody added
Place cover slip
Look under fluorescence microscope, using correct wavelength filter
What are the advantages and disadvantages of immunofluorescence microscopy?
+ ves
Easy to set up
Can clearly localize different organelles, in different colours
- ves
Artefacts may form when fixing to slide
Cells aren’t alive
What are the advantages of using GFP and transmitted light?
Cells can be alive because they don’t have to be fixed
No artefacts
Multicolour can be used
Obtain temporal and spatial information simultaneously
What is tubulin’s role in the cell?
Tubulin polymerises to form microtubules
Involved in the transport of proteins
Important in aligning the chromosomes in the middle in metaphase
Involved in the separation of chromosomes in anaphase
How would you set up a bright field microscope?
1) Start on lowest magnification switch on the light and put into focus using coarse then fine focus
2) Set condenser iris into the middle
3) Close the field iris unti the edges of the hexagon begins to appear
4) Centre the hexagon using the screws
5) Focus the condenser by using the focus knob
6) Open field iris until hexagon about to disappear
7) Take out an eyepiece
8) Adjust the condenser iris until the circle is 2/3 of the heaxgon
9) Replace the eyepiece and observe the slide
Use oil for mag x100
How can resolving power be calculated?
0.61 x wavelength/ NA
Why is it important to measure the amount of chlorophyll in the extracts?
To directly compare rate of photosynthesis, where concentrations may not be the same
Calculate the photosynthetic rate
Calculate the change in mV, per minute
Multiply to change the units to micromol
Divide by the mg of chlorophyll but scaled to how much was present
e.g. 200micro in acetone buffer, but 125 may be used to find the mV readings
What are the advantages and disadvantages of phase contrast?
Adv- Cells can be alive, increased contrast, high resolution
Disadv- can create a halo effect, requires phase rings, limited contrast range
How to prepare cells for TEM?
Gluteraldehyde to fix
dehydrate with acetone
Cut thin section with diamond blade
stain using heavy metal salts
How would you fix and dehydrate a cell?
Fix- gluteraldehyde
Dehydrate- acetone
What is the Beer-lambert law?
A= cel
- A= absorbance
- l= length of cuvette
- e= constant
- c= concentration
What are limitations of the Bradford Assay?
- not very accurate at low conc of protein
- sensitive to protein compositions, some proteins may bind to dye better than others
- can be easily interfered with by buffers, lipids etc.
What is 1 Dalton equal to?
1g/mol
What does high affinity mean in relation to Km?
- low Km is high affinity
What is a start codon for?
To initiate transcription
Code for first amino acid
Regulating accessibility to it, regulates gene expression
What assumptions are being made to obtain the Michaelis-Menten constant?
Reaction carried out under steady state conditions
Enzyme concentration is constant
Initial rate is measured
Propose an experiment to see cell cycle arrest in anaphase
Use fluorescence miscroscopy
- tubulin and DNA
- In anaphase will be as the chromosomes are separating
Propose an experiment to see cell cycle arrest before DNA replication
Immunostain CDKs
In G1 phase, so concentration low
Measure DNA content, extraction and gel electrophoresis or absorbance
Propose an experiment to see cell cycle arrest after DNA replication
Incorporate immunofluorescent tag, which will be added onto new strand of DNA
Monitor location and presence of dye
Give two advantage of Gallus gallus as a model organism.
Vertebrate animal
Evolutionarily conserved
Have large developing embryos, easy to see
Advantages and disadvantages of Arabidopsis
Well known genome (sequenced)
Easy to grow
Small genome, with minimum repeats
Advantages and disadvantages of zebrafish
Transparent embryos
Easy to grow
large number of offspring
- very different to mammals
Advantages and disadvantages of Xenopus
Transparent
Large easy to see and manipulate
Slow generation time
Tetraploid 4 sets of chromosomes
Distant to mammals
Advantages and disadvantages of Drosophila
Short regeneration time
Large number of offspring
Well characterized genome
Transparent embryo
Common model organism, well known genome
Advantages and Disadvantages of C.elegans
Simple organisms
Easy to keep in a lab
Same cell fate
Distant to mammals
Briefly describe why and how cytoplasmic streaming occurs
Occurs for bulk flow movement of organelles
Organelles are attached to a cytoskeleton which causes their movement, actin is polar so moves in one direction
