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Molecular > Labs > Flashcards

Labs Flashcards

1
Q

Restriction enzymes are produced by for
Why not harm the host

A

Prokaryotes
Defense
As the sequence in host is methylated

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2
Q

It cuts
When

A

The phosphodiester bond
When recognising a palindromic seq

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3
Q

The seq is …. Length for restriction site

A

4-6 bases

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4
Q

2 types of nucleases
Naming

A

Endo (inside)
Exo (outside)
Genus, species, strain, recognition number

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5
Q

Star activity
When

A

Losing specificity cuts similar but not identical seq
High ratio of enzymes to DNA
very low ionic strength
Ph
Organic solvents (DMSO- ethanol)
High glycerol conc
Mg2+ substition with other divalents

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6
Q

Cutting types

A

Blunt
Sticky

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7
Q

Applications of restriction enzymes

A

Gene therapy
Genetic engineering
Recombinant proteins
DNA library
DNA fingerprinting
Genome seq
Restriction fragment length polymorphism analysis (diseases )

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8
Q

Restriction enzymes steps

A

Add quantities
Pippette
37 in pcr for 15 mins
5 mins at 80 for enzyme inactivation
On gel

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9
Q

RNA extraction
RNA types

A

mRNA > 7 methyl guanosine head and poly A tail
tRNA> clover like shape
rRNA> non coding, ribosomes (30+50=70)(40+60=80)

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10
Q

Why extract
Genome
mRNA
protein

A

Different alleles, genes presence
Activated genes in tissues, functionality
Misfolding, mRNA expressed or not

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11
Q

Why is it a problem if RNA sample is contaminated with DNA

A

False positive, primers attaching to DNA which does not mean activated genes

Dans are not true results

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12
Q

Ph of RNA. Extraction
DNA

A

4.7
8

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13
Q

Paramagnetic particle tech

A

Lysis buffer and beads with Poly T
Poly T beads attach to Poly A tail
Beads collected by magnet
Rest discarded
Elution buffer
Beads collected
mRNA is left

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14
Q

Another method is for RNA extraction

A

Organic extraction by acid guanidium thiocyanate chloroform
AGPC

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15
Q

Silica membrane based spin column

A

Lysis
Centrifuge to separate debris
Supernatant collected in spin column
Centrifuge
RNA binds to beads with binding buffer
Impurities in collection tube discarded
Ethanol wash
Elution buffer
RNA only

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16
Q

Binding vs elution buffer

A

Binding has high salt conc to form a salt bridge to minimise repulsion bet silica and RNA as they are both negative

Elution has low salt conc to elute RNA from silica

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17
Q

RNA assessment

A

Gel > 3 bands or 2 (28S - 18S - 5S)
Nanodrop 260/ 280 2-2.2
260/230. 2-2.2

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18
Q

RNases

A

Degrade RNA
Omnipotent
Robust
Very destructive

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19
Q

Why are Rnases not eliminated by autoclave and how to eliminate them?

A

As they contain disulphide bonds
By DEPC, histidine specific alkylating agent targets histidine in active site and disrupts it

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20
Q

DEPC is

A

Diethylpyrocarbonate

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21
Q

DEPC steps

A

Add DEPC
RNases inactivated
Autoclave to remove DEPC
RNA suspended in DEPC treated water

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22
Q

Storage

A

-80
Liquid nitrogen
RNA later, conc solution with ammonium and caesium sulphate to denature proteases and RNases

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23
Q

Why extract RNA

A

Real time PCR
next generation seq
Northern blotting
Cloning
Microarray analysis

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24
Q

RNA extraction steps 1

A

Mechanical lysis to liver with liquid nitrogen and mortar and pistol

Chemical lysis with buffer

Chloroform for phases and deproteinisation
Vortex for proteins to be mixed by force
Centrifuge for supernatent to be on silica

Aspire supernatant
Wash with 70 percent ethanol
Spin column
Centrifuge

Removed flow through
Washing buffer
Centrifuge
Washing buffer
Centrifuge

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25
Q

RNA extraction steps 2 after washing buffer

A

Discard flow through
Centrifuge
Collecting tube discarded
Placed in new epindorf

Elution buffer
Centrifuge
Spin column discarded
Storage

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26
Q

Boiling method steps

A

Cell lysis and deproteinisation by high temp then heating to crystalise and denature the proteins

Precipitation by absolute ethanol forms hydration shell, dehydrates

Washing with 70 percent ethanol, 30 water to hydrate and prevent degradation

Dry out to remove excess ethanol

Resuspension in TE buffer or distilled water

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27
Q

Why dry out is important

A

To prevent DNA degredation

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28
Q

DNA precipitation

A

Absolute ethanol, dehydrated (double volume)
Ionic salts, neutralised negative phosphates

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29
Q

TE buffer

A

Tris for pH

EDTA to chelate metal ions as they are cofactors for DNases

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30
Q

Dilution factor is the…of

A

Reciprocal of no. Of times dilutes

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31
Q

Organic extraction

A

Cell lysis by Na2 TRIS-EDTA ans SDS and proteinase K

Deproteinisation by phenol,chloroform,isomamyl and chloroform, isoamyl

Precipitation by absolute ethanol

Washing by 70 percent ethanol

Dry out
Dissolving in TE or distilled water

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32
Q

EFTA is short for
SDS mechanism

A

Ethylene diamine tetra acetic acid

Anionic detergent that dissolves the cell membrane by integrating bet phospholipids and helps to denature proteins and reduce DNase activity

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33
Q

Proteinase k

A

Enzyme to break down proteins and eliminate contaminants
If in eukaryote then histones

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34
Q

Phenol chloroform isoamyl ratio

A

25:24:1

35
Q

Phenol use
Chloroform use
Isoamyl use

A

Phenol denature proteins by exposing the hydrophobic part outside so they will dissolve in it allowing the DNA to separate

Facilitates nucleic acid dissociation from proteins
Removes excess phenol
Increases density to prevent phase inversion

Antifoaming, prevent emulsification

36
Q

Why DNA is on top

A

Cuz phenol and chloroform are denser than water

37
Q

Chloroform is used to rinse…as it is..excess can cause

A

Phenol
Slightly water soluble
Inhibition to enzymatic down stream applications and change in DNA solubility

38
Q

Isopropanol vs ethanol

A

Isopropanol is same volume, ethanol double
Isopropanol is branched so large molecular weight
Isopropanol can be used when there is low DNA conc
Fast at room temp

39
Q

To extract from liver …lysis is needed

A

Mechanical and chemical
Liquid nitrogen and mortar and pistol
SDS and proteinase K has the ability to digest native keratin

40
Q

Phase inversion

A

Organic layer on top and aqueous phase at the bottom if there is high salt conc in buffer

41
Q

Physical lysis

A

Glass beads
Mortar and pistol
Sonication
Tissue homogeniser
Liquid nitrogen = tissue becomes brittle

42
Q

Why use salt when precipitating?

A

As positively charged ions will neutralise negative charges on phosphates so less soluble

43
Q

When to use Hot start DNA pol

A

To reduce non specific amplifications

44
Q

The primer is complementary to…..but it is…

A

3 to 5
5 to3

45
Q

Divalent
Monovalent

A

Cofactors for pol
Minimise repulsion bet primer and template

46
Q

Buffer for PCR has

A

(Nh4)2 SO4
Glycerol
Tris-HCl
KCl

47
Q

Role of glycerol and DMSO

A

Prevent degradation
Relax 2ndry structures with high CG content

48
Q

Why called chain reaction

A

25-45 cycles
Newly amplified DNA copies act as template and number is duplicated

49
Q

PCR steps

A

Initial denaturation
Denaturation
Annealing
Elongation
Final elongation
Holding

50
Q

RAPD PCR stands for
Used to

A

Random amplification of polymorphic DNA
DNA fingerprints of an organism
Differentiate bet species, study phylogeny, estimate genomic variation

51
Q

Concentrations for optimisation
DNA template
Taq
Mg
dNTP
PRIMER

A

50-100ng/50
0.5-2.5 unit/50
1.5mM
220uM
0.1-1uM

52
Q

Low Mgcl2 conc
High

A

No PCR product as low pol activity
Non specific with error

53
Q

Primer length
Short
Long

A

17-24 bp
Non specific
2ndry structures

54
Q

2ndry strictures of primers

A

Hairpin
Self dimer (forward and forward)
Cross dimer (forward and reverse)

55
Q

Very high GC content

A

2ndry structures
High temp req so extension will start with anealing

56
Q

Equation for melting temp
Anealing

A

4(C+G)+ 2(A+T)
Melting - 5

57
Q

Dont do in primers

A

No complementary ends
No self complementary
3 or more C or G at the 3’ end

58
Q

Do we calculate the melting temp alone for each primer, which temp
Melting temp range

A

Yes
Forward and reverse calculated alone and the lowest temp is taken for it not to be the melting temp of the other
55-65

59
Q

Do we use a stain with cosmo red PCR mix when running on gel

A

No

60
Q

How to troubleshoot

A

Identify problem
Solve it

61
Q

Methods for DNA extraction
Lysis buffer contains

DNA precipitation with abs ethanol needs

A

Organic
Boiling
Chelex
Solid phase
Tris-EDTA and SDS and proteinase K

Ice for 15 mins

62
Q

Concentration of DNA equation
Yield

A

(A260-A320)50dilution factor
Concentration* sample volume

63
Q

Why DNA absorbs light strongly at A260
230nm what will be detected
Range of pp
Range of cp

A

Due to the heterocyclic rings
Phenols, guanidine, carbs
1.7-2
2-2.2

64
Q

Steps for agarose gel

A

Percentage selection
Running and sample buffet
Voltage and runtime calc
Gel recipe and DNA staining visualisation

65
Q

Running buffer func
Types

A

Conduct current
Resist pH changes
TAE, tris- acetic acid- EDTA
TBE, tris- boric acid- EDTA
faster better

66
Q

Running buffers compare

A

TAE is cheap, fast and for large fragments
TBE is expensive slow for long runs and can be reused for small fragments and more stable
Faster better, less running time tolerates up to 275V

67
Q

Sample buffer conatins

A

Glycerol / sucrose/ficoll 400 and tracking dye like bromophenolblue , orange G, xylene cyanol
TRIS

68
Q

Voltage calc

A

4-8 volt per cm from cathode to anode

69
Q

Ethidium bromide used to….
Works by ..to..

A

Stain DNA
Intercalating into the DNA molecule and forming close van der waal forces
Flourescence

70
Q

Intensity of band is prop to
Integrity

A

Conc
Intact or smeary

71
Q

Why running buffer should cover the whole sample
How to view ethidium bromide

A

For effective conductivity
Under UV

72
Q

Other stains for viewing the gel

A

Ethidium bromide for DNA
SYBR GOLD
SYBER GREEN proteins
crystal violet and methyl blue do not need UV
SYBER safe
Green safe
GEL STAR safe

73
Q

Gel
Smeared bands
Cloud down

A

DNA degraded due to low TRIS/ high DNA conc/ protein contamination/ improper electrophoresis / small pore size/ high voltage

RNA contamination

74
Q

70 % ethanol why
Salt for preciptation

A

70 to remove excess salt
30 to hydrate
Neutralisation of negative charges on phosphate

75
Q

Resuspension in TE or DW for long term

A

TE DUE TO EDTA

76
Q

Tracking dye should be

A

Light than sample

77
Q

Case study
Nth happened after 30 mins of gel electrophoresis

Loaded sample then loading buffer

A

No conduction running buffer not covering the sample, add more running buffer

Sample will float

78
Q

Which stain to use if I want to see a PCR product that will be cloned

A

Any stain that does not need UV
crystal violet or methylene blue

79
Q

Gel empty under UV
Ladder overlapping
White cloud down
All bands up
Cloud up

A

Problem in visualising stain
Volt or gel problem
High volt/ RNA contamination
Little buffer
Smearing

80
Q

Line up and cloud down in DNA assessment vs PCR

A

Rna contamination
Primer dimer

81
Q

Ratio sample buffer to DNA

A

1:5

82
Q

Band up in RNA gel

A

DNA, needs DNase treatment
Wrong pH was used

83
Q

No band

A

High contamination
No denaturation
Low divalent
High annealing temp
Low purity

84
Q

Non specific bands

A

Low annealing
High Mg2
Use hot start

Molecular (6 decks)

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