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Practicals Flashcards

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1
Q

State important design features of a primer

A

3’ CG clamp
20bp minimum length
Equal distribution of A,T,C,G
No palindromes
No homopolymeric sequences
No self complementary regions

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2
Q

How is the melting temperature (Tm) calculated?

A

Wilson’s Rule
2(A+T)+ 4(C+G)

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3
Q

How is the annealing temperature calculated?

A

-2 degrees from melting temperature

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4
Q

What is the reason for 94, 48 and 72 degrees being used in PCR?

A

94 denaturation of DNA strands
48 annealing temperature
72 primer extension, replication temp

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5
Q

How does ethidium bromide allow you to visualize a gel electrophoresis?

A

It intercalates between DNA bases
When bound
Becomes fluorescent under UV

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6
Q

How to calculate the amount of product produced from a PCR?

A

1) Calculate efficiency
2) Multiply by 2^ (cycles)

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7
Q

Suggest one simple strategy by which mis-priming can be alleviated

A

Hot-start
Increase annealing temperature
(If Mg2+ used, decrease conc of Mg2+, because it is present to stabilize DNA)

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8
Q

What is the definition of spatial resolving power?

A

Ability to distinguish between two closely located objects as two separate entities

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9
Q

How can RNA and DNA be stained?

A

RNA- Pyronin (pink)
DNA- DAPI (blue)

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10
Q

What is a somite?

A

Cells that give rise to ribs and spine
Drosophila (synchronous segmentation)
Chick (sequential segmentation)

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11
Q

How do you determine the single trait ratio?

A

+/-

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12
Q

How would you test if a mutation is in a single gene?

A

Backcross mutant with WT
expect a 1:1

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13
Q

How would you test whether a mutation is on the same gene or not?

A

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

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14
Q

What is epistasis and how does it differ from dominance?

A

epistasis- masking of one gene by another
Dominance- masking of an allele by another, but on the same gene

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15
Q

What aspects of the Aspergillus nidulans (fungus) life cycle make it a useful model organism for the genetic analysis of a metabolic pathway?

A

Form heterokaryons
Haploid so don’t show dominance
Produces many progeny
Easy to grow

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16
Q

How would you test whether a mutation in Saccharomyces cerevisiae is derived from a mutation in the nuclear gene or a mitochondrial gene?

A

Cross with WT
1:1 is nuclear
Uneven distribution if mitochondrial

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17
Q

What is the Shine- Dalgarno sequence?

A

Ribosome binding sequence
Located 8bp before AUG start codon
Helps recruit ribosome

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18
Q

How to tell whether the gene will be efficiently transcribed?

A

If the sequence doesn’t differ much from the consensus sequence

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19
Q

Why is ice cold Calcium chloride used in the transformation of bacteria?

A

Speed up the process of plasmid transformation
cold shock required
Overall increasing efficiency

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20
Q

What control would need to be implemented to show that the results are due to the plasmids and not by contamination?

A

Carry out same experiment in another tube
However don’t add plasmids to that tube

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21
Q

What is the purpose of rapid heating and cooling of the lysate, in plasmid extraction?

A

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

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22
Q

Why is there a high concentration of mannitol in the buffer?

A

Acts as an isotonic buffer
Stop osmotic lysis

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23
Q

What does pKa mean?

A

How acidic an atom of H is
-logKa
Low pKa= strong acid

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24
Q

Describe in detail the method of staining pancreatic acinar cells

A

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

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25
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
26
How are bird RBC different to human RBC?
Bird RBC have a nucleus
27
How would you estimate the size of a cell?
use graticule Know the dimensions that correlate to the magnification
28
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
29
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
30
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
31
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
32
An area of protein has no secondary structure suggest why
Gives protein flexibility If close to ligand binding site Allows for conformational change
33
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
34
Explain how you would distinguish between a male and a female drosophila
Male- dark abdomen Female - lighter abdomen, usually larger also
35
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
36
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
37
What is melting temperature?
When 50% of primers are bound and 50% are unbound
38
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
39
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
40
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
41
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
42
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
43
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
44
How can resolving power be calculated?
0.61 x wavelength/ NA
45
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
46
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
47
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
48
How to prepare cells for TEM?
Gluteraldehyde to fix dehydrate with acetone Cut thin section with diamond blade stain using heavy metal salts
49
How would you fix and dehydrate a cell?
Fix- gluteraldehyde Dehydrate- acetone
50
What is the Beer-lambert law?
A= cel - A= absorbance - l= length of cuvette - e= constant - c= concentration
51
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.
52
What is 1 Dalton equal to?
1g/mol
53
What does high affinity mean in relation to Km?
- low Km is high affinity
54
What is a start codon for?
To initiate transcription Code for first amino acid Regulating accessibility to it, regulates gene expression
55
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
56
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
57
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
58
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
59
Give two advantage of Gallus gallus as a model organism.
Vertebrate animal Evolutionarily conserved Have large developing embryos, easy to see
60
Advantages and disadvantages of Arabidopsis
Well known genome (sequenced) Easy to grow Small genome, with minimum repeats
61
Advantages and disadvantages of zebrafish
Transparent embryos Easy to grow large number of offspring - very different to mammals
62
Advantages and disadvantages of Xenopus
Transparent Large easy to see and manipulate Slow generation time Tetraploid 4 sets of chromosomes Distant to mammals
63
Advantages and disadvantages of Drosophila
Short regeneration time Large number of offspring Well characterized genome Transparent embryo Common model organism, well known genome
64
Advantages and Disadvantages of C.elegans
Simple organisms Easy to keep in a lab Same cell fate Distant to mammals
65
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

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