Practical Molecular Bioscience Flashcards
(92 cards)
1
Q
What is % (w/v)?
A
- g/100ml
2
Q
What is M?
A
- mol/l
3
Q
What is accuracy?
A
- whether all values are close to true value
4
Q
What is consistency (precision)?
A
- whether all values are close to each other
5
Q
Why would you dilate bacteria in saline rather than water?
A
- saline has same Ψ as bacterial cells
- pure water would cause water to enter cells and burst
6
Q
What is the difference between viable cell count and total cell count?
A
- viable is actively growing cells
- total inc dead cells
7
Q
What are the types of microbial media?
A
solid:
- slope culture
- stab culture
- plate culture
- streak plate culture
- spread plate culture
- pour plate culture
- overlay culture
liquid:
- broth culture
8
Q
What is a slope culture and when is it appropriate to use?
A
- tubes containing ~10ml agar or gelatin medium heated to melt medium
- allowed to set in sloping position
- increasing surface area
- inoculum spread over surface or applied in thin streak w/ sterile wire group
- approp for culturing small amounts of bacteria
9
Q
What is a stab culture?
A
- tubes containing ~10ml agar medium, allowed to solidify in upright position
- inoculated by straight needle plunged vertically deep into medium
10
Q
What is a plate culture?
A
- ~20ml solid agar medium for each petri dish
- medium melted and cooled to 50ºC
11
Q
What is a streak plate culture and what does it allow?
A
- cooled medium poured into petri dish and allowed to set in smooth even layer
- inoculum streaked onto surface of medium to prod well separated colonies after incubation
- allows cells to be spread more evenly , so individual colonies can be isolated
12
Q
What is a spread plate culture?
A
- plate prepared like streak
- inoculum is drop of liquid bacterial culture
- transferred w/ pipette and spread using spreader dipped in alcohol and passed through bunsen flame (sterilising it)
13
Q
What is a pour plate culture, and when is it a useful technique?
A
- inoculum added to melted and cooled agar medium
- gently mixed and poured into plates
- or inoculum placed into petri dish and liquid medium added
- to ensure even distribution, moved w/ rotating movement
- useful for isolation of bacterial cultures from mixed inoculum, or where heavy even growth needed
14
Q
What is an overlay culture?
A
- similar to pour plates
- inoculum mixed w/ smaller amount of melted agar medium
- poured in thin layer to solidify on top of solid medium already in petri dish
15
Q
What is a broth culture and what does it produce?
A
- inoculum transferred using sterile wire loop into liquid medium
- produces large no.s bacteria, but not discrete colonies
16
Q
What are stages of a gram stain?
A
- heat fixed smear
- stain w/ methyl violet
- iodine to trap stain in peptidoglycan layer
- ethanol wash
17
Q
What do the results of a gram stain indicate about whether cells are gram +ve or -ve?
A
- +ve = purple
- -ve = pink/red
18
Q
What is the difference between gram +ve and gram -ve cells?
A
- gram +ve have thick cell wall, so retains methyl violet
- gram -ve have thin cell wall
19
Q
What is the appearance of Staphylococcus aureus on solid media, its morphology and gram result?
A
- yellow
- clusters of cocci
- +ve
20
Q
What is the appearance of E. coli on solid media, its morphology and gram result?
A
- white-grey
- individual rods
- -ve
21
Q
What is the appearance of B. megaterium on solid media, its morphology and gram result?
A
- large white colonies w/ diffuse edges
- rods, short chains
- +ve
22
Q
What is the appearance of Serratia marcescens on solid media, its morphology and gram result?
A
- red
- short rods
- -ve
23
Q
What is the appearance of Streptococcus agalactiae on solid media, its morphology and gram result?
A
- white-grey
- cocci, short chains
- +ve
24
Q
Why are chromosomes not visible under the light microscope in interphase?
A
- in highly diffuse and extended form
25
How is prophase visible?
- chromosomes become visible due to progressive shortening and thickening
- at end begin to move towards spindle equator (prometaphase)
26
How is metaphase visible?
- chromosomes arranged w/ centromere attached to equator
27
How is anaphase visible?
- centromeres split and chromosomes move to opposite poles
28
How is telophase visible?
- 2 groups of chromosomes decondense and daughter nuclei form
29
In what cells are the stages of mitosis easily observable?
- root tips (actively growing)
30
What does metacentric mean?
- centromere in middle
31
What does submetacentric mean?
- centromere between middle and end
32
What does acrocentric mean?
- centromere near 1 end
33
What does telocentric mean?
- centromere at 1 end
34
What does G banding do?
- stains gene-poor, heterochromatic regions
- leaving transcriptionally active regions unstained
- generates specific banding pattern for each chromosome
35
Would you expect standard curve for spectrophotometer to be linear and how long for?
- in theory always linear
- in practise it wouldn't, as machine can't accurately measure v small amount of light being transmitted
- compound becomes more conc, will reach point where no longer be soluble
36
What does absorbance equal?
- A = ECt
- E is extinction coefficient
- C is conc
- t is path length
37
How is pH calculated from [H+]?
-log[H+]
38
What is the equation for Ka?
Ka = [H+][A-] / [HA]
39
What is the Henderson-Hasselbalch equation?
pH = pKa + log( [A-] / [HA] )
40
How is pKa calculated from [Ka]?
-log[Ka]
41
What is the relationship between pH and pKa when [A-] = [HA]?
- pH = pKa
42
What are the limitations of Lineweaver-Burk plot?
- difficult to find x-intercept as never any minus values
- bunching of lower values relied on to draw line to bigger values
- larger values unreliable as at low substrate conc
43
What are the characteristics req of enzymes in biological washing powder?
- tolerate high temp and high activity at low temps
| - tolerate wide range of pHs
44
What sort of enzymes are present in biological washing powders, and what do they do?
- proteases
| - hydrolyse proteinaceous stains
45
What is an autoclave?
- strong heated container used to sterilise equipment by subjecting them to high pressure steam at 138kPa, heated to 121º for 20 mins
46
What are sulphatases?
- enzymes that hydrolyse sulphuric acid esters
47
How can optical density be used to measure bacterial growth?
- cells in suspension scatter light
- only proportion of light passes through to be detected by photocell in spectrophotometer
- as cell density increases w/ growth, more light scattered and optical density increases
48
How are biochemical assays carried out to count cells?
- cells collected by centrifugation, washed, dried in oven and weighed
49
What are the 5 stages of prophase I in meiosis and what occurs in each?
- leptotene --> condensation begins, paired chromatids, extended thread-like structure
- zygotene --> assoc in bivalents by synapsis
- pachytene --> shorter and thicker, crossing over but not visibly detectable
- diplotene --> 2 homologous chromosomes w/in each bivalent separate over most of length inc centromeres, held together at chiasmata, sister chromatids remain attached at centromeres
- diakinesis --> further shortening and thickening
50
When are temp sensitive mutants useful?
- when identifying genes that are essential for survival
51
What is epistasis?
- occurs when allele of 1 gene masks expression of either allele of a 2nd gene
52
What is complementation and when does it occur?
- effects of 2 diff non-allelic mutations in gene partly/completely cancelled out when occur together
- only occurs if mutant alleles recessive to WT
53
What are allelic and non-allelic mutants? (complementation)
- allelic mutants carry mutants in some gene
| - non-allelic mutants carry mutations in diff gene (same phenotype)
54
What features do plasmids used as vectors generally have?
- contain origin of replication functional in E. coli
- multiple restriction sites, which can be used to insert foreign DNA
- encode gene for resistance for antibiotic as means of selection
- relatively small and present at high copy no. per cell so can be purified in large amounts
55
What are the diff topological species that can be present of plasmid DNA molecules?
- supercoiled
- circular nicked
- linear
- supercoiled dimer
56
What do the no.s of diff topological species differ depending on? (plasmid DNA molecules)
- copy no. of vector
- size of vector
- length of time plasmid exposed to alkaline conditions
57
At what rate do linear ds DNA molecules migrate through electrophoresis?
- rate inversely proportional to log of their molecular weight
58
How does the topology of plasmid DNA molecules affect migration through the gel?
- small plasmids mainly isolated as monomeric supercoiled form
- large plasmids often exclusively nicked and rarely isolated in supercoiled form
- generally more compact molecules will migrate faster through gel --> supercoiled faster than nicked, faster than linear
59
When can sizing of plasmid be carried out?
- when converted to linear molecule
| - digestion w/ restriction endonuclease, w/ single recognition site w/in plasmid seq
60
What % agarose gels do labs typically use and what can this resolve?
- 1%
| - can adequately resolve DNA fragments ~1-10kb
61
What effects do lactose, IPTG and ONPG have on the lac operon?
- lactose --> converted to allolactose which binds to lac repressor, broken down by β-galactosidase to glucose and galactose
- IPTG --> binds to lac repressor, but not broken down by β-galactosidase
- ONPG --> doesn't bind to lac repressor, broken down by β-galactosidase to galactose and ο-nitrophenol, prod yellow colour
62
What are the stages of gene cloning experiment?
- preparing vector and insert molecule, to be joined
- joining DNA fragments = ligation
- introducing ligated molecule into host organism = transformation
63
What is the only topology of plasmid which can be replicated and confer antibiotic resistance on bacteria, and why?
- circular
| - linear quickly degraded by bacterial enzymes
64
What are the different methods of transformation? (gene cloning experiment)
- preparation of competent cells
- transformation by heat shock
- antibiotic selection
65
What factors can proteins be purified by?
- size
- shape
- charge
- solubility
- stability
- binding specificity
66
How does gel filtration work?
- molecules fractionated according to size (and independently of charge)
- as they pass through column packed w/ gel matrix
- matrix consists of porous beads filled w/ liquid
- as proteins flow past, can diffuse into internal pore structure to extent based on size
- smaller molecules diffuse further into pores, so move through column slower
- molecules too big to enter pores at all excluded from beads, so only pass through solution surrounding beads and elute first
67
What is void volume (Vo)? (gel filtration)
- volume of elution of molecules excluded from beads equal to volume of liquid outside beads
68
What is elution volume (Ve)? (gel filtration)
- molecules of different sizes that can enter beads eluted from column w/ specific Ve
69
What is VI? (gel filtration)
- whole volume of liquid in column
| - smallest molecules that can completely penetrate beads elute w/ volumer equal to Vl
70
What is Vi and how is it calculated?
- volume of liquid inside beads
| - Vi = Vl - Vo
71
What is Vt and how is it calculated?
- total column volume
| - Vt = Vo + Vi + Vs
72
What is Vs?
- volume of solid beads
| - usually between 5 and 15%
73
How can molecules of different sizes be excluded in gel filtration?
- extent of cross-linking, determines pore size in beads
| - smaller pore size = higher level cross linking
74
What are the practical uses of gel filtration?
- desalting or buffer exchange --> v small pores, excludes most proteins but allows salts to enter pores, used whn protein needs to be salt free or in diff buffer conditions for subsequent experiment
- purification of macromolecules --> separating diff sizes
- analysis of oligomeric state of proteins and protein complexes --> column can be calibrated using proteins of known sizes, then used to determine protein size
75
What are the differences between cation and anion exchange chromatography?
- cation = purify acidic proteins, has -vely charged ions, so attracts +ve proteins
- anion = purify basic proteins, has +vely charged ions, so attracts -ve proteins
76
What effect does high salt conc have on proteins bound to ion exchange resin, and why?
- ions in salt compete w/ protein to bind to resin
- as they bind to +ve/-ve charged groups on resin
- salt in great excess
77
What is apyrase and why is it used?
- has ADPase and ATPase activity
| - ensures any residual ADP and ATP removed
78
How could respiratory activity be measured?
- measure O2 uptake
| - use different acceptor in place of O2, w/ colour change when red, eg. DCPIP
79
What properties of Dunaliella make them suitable for growth in outdoor ponds?
- photosynthetic
| - motile
80
What are the main products that can be synthesised from Dunaliella?
- vegetable oil
- food colourings
- aquaculture
81
What are the components of Laemmli buffer?
- 10% (w/v) SDS
- glycerol
- 1M Tris-Cl
- water
- bromophenol blue
82
What is the purpose of Laemmli buffer?
- reduces disulfide bonds of proteins to allow proper separation by size, not shape
83
What are 2 alternatives to overexpression of bacterial proteins in bacteria?
- overexpression of recombinant proteins in eukaryotic cells
| - in vitro translation of recombinant proteins
84
How can you check cloning has worked correctly?
- restriction digest to quickly check insert in correct orientation
- sequencing to see if any mutations in insert seq (perhaps introd by PCR)
85
How does SDS-PAGE work?
- SDS denatures proteins, so run on gel according to molecular weight not structure
86
What is the formula for chi-squared?
Σ { (O-E)2 / E }
87
How is dilution factor calculated?
- total final vol / vol of original solution
| - starting conc / final conc
88
How is total dilution factor of serial dilutions calculated?
- multiply individual dilution factors together
89
What are doubling dilutions?
- series of 2 fold dilutions
90
What are the prefixes for the different powers of 10 from 6 to -15?
- 6 = mega (M)
- 3 = kilo (k)
- 2 = hecto (h)
- 1 = Deca (da)
- -1 = deci (d)
- -2 = centi (c)
- -3 = milli (m)
- -6 = micro (μ)
- -9 = nano (n)
- -12 = pico (p)
- -15 = femto (f)
91
How can volumes in L be converted to volumes in m^3?
- 1L = 1dm^3
- 1ml = 1cm^3
- 1μl = 1mm^3
92
How do you calculate standard deviation?
√ of Σ {(x – x)^2} / (n-1)
