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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
RNA extraction steps 2 after washing buffer
Discard flow through Centrifuge Collecting tube discarded Placed in new epindorf Elution buffer Centrifuge Spin column discarded Storage
26
Boiling method steps
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
27
Why dry out is important
To prevent DNA degredation
28
DNA precipitation
Absolute ethanol, dehydrated (double volume) Ionic salts, neutralised negative phosphates
29
TE buffer
Tris for pH EDTA to chelate metal ions as they are cofactors for DNases
30
Dilution factor is the...of
Reciprocal of no. Of times dilutes
31
Organic extraction
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
32
EFTA is short for SDS mechanism
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
33
Proteinase k
Enzyme to break down proteins and eliminate contaminants If in eukaryote then histones
34
Phenol chloroform isoamyl ratio
25:24:1
35
Phenol use Chloroform use Isoamyl use
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
Why DNA is on top
Cuz phenol and chloroform are denser than water
37
Chloroform is used to rinse...as it is..excess can cause
Phenol Slightly water soluble Inhibition to enzymatic down stream applications and change in DNA solubility
38
Isopropanol vs ethanol
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
To extract from liver ...lysis is needed
Mechanical and chemical Liquid nitrogen and mortar and pistol SDS and proteinase K has the ability to digest native keratin
40
Phase inversion
Organic layer on top and aqueous phase at the bottom if there is high salt conc in buffer
41
Physical lysis
Glass beads Mortar and pistol Sonication Tissue homogeniser Liquid nitrogen = tissue becomes brittle
42
Why use salt when precipitating?
As positively charged ions will neutralise negative charges on phosphates so less soluble
43
When to use Hot start DNA pol
To reduce non specific amplifications
44
The primer is complementary to.....but it is...
3 to 5 5 to3
45
Divalent Monovalent
Cofactors for pol Minimise repulsion bet primer and template
46
Buffer for PCR has
(Nh4)2 SO4 Glycerol Tris-HCl KCl
47
Role of glycerol and DMSO
Prevent degradation Relax 2ndry structures with high CG content
48
Why called chain reaction
25-45 cycles Newly amplified DNA copies act as template and number is duplicated
49
PCR steps
Initial denaturation Denaturation Annealing Elongation Final elongation Holding
50
RAPD PCR stands for Used to
Random amplification of polymorphic DNA DNA fingerprints of an organism Differentiate bet species, study phylogeny, estimate genomic variation
51
Concentrations for optimisation DNA template Taq Mg dNTP PRIMER
50-100ng/50 0.5-2.5 unit/50 1.5mM 220uM 0.1-1uM
52
Low Mgcl2 conc High
No PCR product as low pol activity Non specific with error
53
Primer length Short Long
17-24 bp Non specific 2ndry structures
54
2ndry strictures of primers
Hairpin Self dimer (forward and forward) Cross dimer (forward and reverse)
55
Very high GC content
2ndry structures High temp req so extension will start with anealing
56
Equation for melting temp Anealing
4(C+G)+ 2(A+T) Melting - 5
57
Dont do in primers
No complementary ends No self complementary 3 or more C or G at the 3' end
58
Do we calculate the melting temp alone for each primer, which temp Melting temp range
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
Do we use a stain with cosmo red PCR mix when running on gel
No
60
How to troubleshoot
Identify problem Solve it
61
Methods for DNA extraction Lysis buffer contains DNA precipitation with abs ethanol needs
Organic Boiling Chelex Solid phase Tris-EDTA and SDS and proteinase K Ice for 15 mins
62
Concentration of DNA equation Yield
(A260-A320)*50*dilution factor Concentration* sample volume
63
Why DNA absorbs light strongly at A260 230nm what will be detected Range of pp Range of cp
Due to the heterocyclic rings Phenols, guanidine, carbs 1.7-2 2-2.2
64
Steps for agarose gel
Percentage selection Running and sample buffet Voltage and runtime calc Gel recipe and DNA staining visualisation
65
Running buffer func Types
Conduct current Resist pH changes TAE, tris- acetic acid- EDTA TBE, tris- boric acid- EDTA faster better
66
Running buffers compare
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
Sample buffer conatins
Glycerol / sucrose/ficoll 400 and tracking dye like bromophenolblue , orange G, xylene cyanol TRIS
68
Voltage calc
4-8 volt per cm from cathode to anode
69
Ethidium bromide used to.... Works by ..to..
Stain DNA Intercalating into the DNA molecule and forming close van der waal forces Flourescence
70
Intensity of band is prop to Integrity
Conc Intact or smeary
71
Why running buffer should cover the whole sample How to view ethidium bromide
For effective conductivity Under UV
72
Other stains for viewing the gel
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
Gel Smeared bands Cloud down
DNA degraded due to low TRIS/ high DNA conc/ protein contamination/ improper electrophoresis / small pore size/ high voltage RNA contamination
74
70 % ethanol why Salt for preciptation
70 to remove excess salt 30 to hydrate Neutralisation of negative charges on phosphate
75
Resuspension in TE or DW for long term
TE DUE TO EDTA
76
Tracking dye should be
Light than sample
77
Case study Nth happened after 30 mins of gel electrophoresis Loaded sample then loading buffer
No conduction running buffer not covering the sample, add more running buffer Sample will float
78
Which stain to use if I want to see a PCR product that will be cloned
Any stain that does not need UV crystal violet or methylene blue
79
Gel empty under UV Ladder overlapping White cloud down All bands up Cloud up
Problem in visualising stain Volt or gel problem High volt/ RNA contamination Little buffer Smearing
80
Line up and cloud down in DNA assessment vs PCR
Rna contamination Primer dimer
81
Ratio sample buffer to DNA
1:5
82
Band up in RNA gel
DNA, needs DNase treatment Wrong pH was used
83
No band
High contamination No denaturation Low divalent High annealing temp Low purity
84
Non specific bands
Low annealing High Mg2 Use hot start

Molecular (6 decks)

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