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PHA111 > Working With Nucleic Acids + Proteins > Flashcards

Working With Nucleic Acids + Proteins Flashcards

1
Q

what do you need to think about when working with cells + tissues?

A
  • temp effects
  • microbial contamination
  • changes in sample content (metabolic reactions/breakdown)
  • is container/bag changing the sample
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2
Q

describe cell isolation?

A
  • remove culture supernatant + add pre-warmed trypsin EDTA to cell monolayer
  • add equal vol of complete medium
  • to neutralise trypsin + collect cells in tube
  • centrifuge at 200 x for 3 mins to pellet cells
  • remover supernatant
  • plate cells once resuspended with culture medium into culture vessels
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3
Q

what is trypsin?

A

enzyme that breaks down proteins

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4
Q

what is EDTA?

A

chemical that chelates/removes divalent cations

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5
Q

describe cell transformation/engineering?

A
  • DNA/RNA + transfection reagent
  • incubate for 20 mins
  • transfection complex added to cells
  • assay cells 24-72 hours post transfection
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6
Q

describe differential centrifugation?

A
  • heterogenous sample added to vessel
  • sample separated via centrifugal force
  • sample separated into pellet and supernatant sample
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7
Q

what can differential centrifugation be used to separate?

A
  • cells from cell growth media

- precipitates from soluble fractions

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8
Q

describe rate-zonal centrifugation?

A
  • heterogenous sample added to vessel with sedimentation gradient matrix
  • sample separated via centrifugal force - into discrete zones of tube/matrix
  • sample separated into sediment fractions + particles isolated from each fraction
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9
Q

what can rate-zonal centrifugation be used to isolate?

A
  • cell structures/organelles

- proteins/rna/dna based on shape/size

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10
Q

cell lysis techniques

A
  • purify proteins to disrupt tissues + cells
  • homogenise plasma membrane to rupture cells
  • homogenate has large and small mol from cytosol e.g. enzymes, ribosomes etc
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11
Q

what are the 4 common procedures used to rupture the plasma membrane of cells?

A
  • break cells with high frequency sound
  • use mild detergent to make holes in membrane
  • force cells through small hole using high pressure
  • shear cells between close fitting rotating plunger + thick walls of glass vessel
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12
Q

what are the physiochemical properties of proteins?

A
  • contain C, H, O, N
  • have defined structure/shape
  • have defined mass
  • absorb light in UV range
  • have both charged + uncharge AA
  • have hydrophilic + hydrophobic AA
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13
Q

proteins in a solution absorb UV light with an absorbance maximum of 280nm

what is the reason for this?

A

due to the aromatic AA

phenylalanine, tryptophan, tyrosine, histidine

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14
Q

what is A280 (UV absorption) used for?

A

detect proteins during purification

routinely used to detect proteins on chromatographic separation

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15
Q

what law can you use to determine protein concentration?

A

beer Lambert law

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16
Q

describe the action of large proteins in size exclusion chromatography?

A

pass through column first

as have shorter flow path through column

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17
Q

in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis, what is the gel?

A

porous mesh of acrylamide fibres

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18
Q

what are proteins given in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis?

A

overall net neg charge by coating them in SDS detergent

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19
Q

describe the action of proteins in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis/

A
  • large proteins find it difficult to move through mesh —> take longer
  • smaller proteins move more quickly
  • reducing agents added to sample to break S-S bonds - unlink different PPC
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20
Q

the electrophoretic mobility of a protein on SDS polyacrylamide gel is related to its…

A

Mr

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21
Q

western blotting

A
  • used detect specific proteins after SDS PAGE separation
  • using antibodies to proteins of interest
  • proteins transferred to membrane
  • membrane probed for specific proteins with antibodies (usually carrying chemiluminescent tag)
22
Q

what are the different types of chromatography?

A
  • gel filtration
  • antibody-affinity
  • ion-exchange
23
Q

for the Edman degradation, after how many AA can you usually identify the protein?

A

10

24
Q

where does the stability of nucleic acids come from?

A

stacking interactions + water exclusion from internal bases

25
Q

when do nucleic acids become hydrolysed completely to their constituent bases?

A

in strong acidic conditions
(HClO4)

more than 100 degrees

26
Q

when does a nucleic acid become apurinic?

A

in more dilute mineral acid

e.g ph 3-4

most easily hydrolysed bonds = selectively broken

27
Q

increasing the ph above the physiological range causes a change in…

A

tautomeric state of bases

28
Q

at neutral ph, the compound is predominantly in what form?

A

keto form

29
Q

increasing the ph will cause a shift to what form?

A

enolate form

30
Q

what is the enolate form?

A

when mol loses proton

since neg charge = most stably accommodated on electroneg oxygen atom

31
Q

in rna, the denaturation of helical regions takes place at a higher ph

there is also a susceptibility of rna to hydrolysis

how so?

A

comes about because of presence of 2 OH group in RNA

32
Q

the cleavage of RNA back-bone is via…

A

intramolecular attack on phosphate of phosphodiester bond

reaction is promoted by high pH, since OH acts as general base

33
Q

the products of rna hydrolysis are…

A

free 5-OH
2,3-cyclic phosphodiester

subsequently hydrolysed to 2 or 3 monophosphate

34
Q

dsDNA is..

A

hypochromic

35
Q

ssDNA is…

A

hyperchromic

36
Q

how can the purity of nucleic acid duplexes be estimated?

A

determining the ratio of absorbance at 260 and 280nm

37
Q

pure dna has a ratio of..

A

1.8

38
Q

pure rna has a ratio of…

A

2.0

39
Q

protein will have a ratio of…

A

<1

40
Q

the length of a nucleic acid will affect the..

A

extinction coefficient

41
Q

in ssDNA and RNA, the base composition affects the..

A

absorbance

purines have higher absorbance than pyrimidines at 260nm

42
Q

the melting of a duplex is the result of..

A

disruption of electronic interactions in the duplex

43
Q

for dna, which regions will denature first and induce the cooperative denaturation?

A

A-T regions

44
Q

what is the effect of chaotrope + solvent addition on nucleic acids?

A

cause denaturation

caused by disruption of HB

45
Q

the incorporation of … will stop the replication process in the Sanger method

A

dideoxynucleotide

46
Q

what do you need for the Sanger method?

A
  • dna to = sequenced
  • dna polymerase
  • primer
  • four dNTPs
  • small amount of ddNTP
47
Q

what are phosphoramidites?

A

modified nucleosides

stander chemical used in modern DNA synthesis

efficient in cyclic reactions

48
Q

synthesising dna process

A
  • nitrogenous bases need = protected
  • immobilise first nucleoside to silica support
  • deprotect sugar - remove DMT
  • couple next protected nucleoside
  • oxidation of phosphate group + cycle repeats
  • remove nucleic acid from silica support
  • remove remaining protecting groups
49
Q

why does thymine not need protecting?

A

has free NH2

50
Q

dna can be cut into sequence specific manner by…

A

restriction endonuclease

51
Q

what do restriction endonucleases do?

A

break phosphodiester bonds in both strands to generate sticky/blunt ends

PHA111 (30 decks)

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