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Summary of DNA modification period 2.1
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Summary DNA modificationAlgemeen doel van de cursus
“De student kan de juiste recombinante DNA-technieken selecteren en aanpassen aan de
hand van een research question, zodanig dat de student een strategie uit kan denken om
kloneringen uit te voeren op het lab.”
Inhoudsopgave
Algemeen doel van de cursus ........................................................................................................................... 1
Les 1 ................................................................................................................................................................. 2
Je kan de werking van verschillende reagentia en chemicaliën die in isolatieprotocollen worden gebruikt voor
plasmide, RNA en genomisch DNA beschrijven. ................................................................................................. 2
Lysate ............................................................................................................................................................. 2
gDNA .............................................................................................................................................................. 3
Overview:....................................................................................................................................................... 4
Solid-phase extraction ................................................................................................................................... 5
Organic extraction ......................................................................................................................................... 6
Salting out ...................................................................................................................................................... 7
Overview ........................................................................................................................................................ 7
Limits of each technique................................................................................................................................ 8
Je kan de elementen van een cloning vector beschrijven en de elementen samenstellen aan de hand van een
research question. ............................................................................................................................................ 12
gDNA extracten plant: basics ......................................................................................................................... 8
Overview:....................................................................................................................................................... 9
DNA extraction from plants ........................................................................................................................... 9
Plasmid isolation .......................................................................................................................................... 10
Je kan de elementen die een rol spelen bij transcriptie en translatie in een eukaryoot en prokaryoot
modelsysteem benoemen en hun functie beschrijven. ..................................................................................... 11
RNA isolation (organic extraction) ............................................................................................................... 11
Reverse transcriptase .................................................................................................................................. 12
Cloning vector utilities: basics ..................................................................................................................... 12
Elements for gene / protein expression ...................................................................................................... 13
ORF versus CDS ............................................................................................................................................ 13
Selecting the right promoter ....................................................................................................................... 14
Inducible promoter ...................................................................................................................................... 14
Blue-white screening ................................................................................................................................... 15
bacteriophage promoter (DE3).................................................................................................................... 16
Choice of vector depends on research question ......................................................................................... 16
Reporter genes ............................................................................................................................................ 17
Transcriptional versus translational fusion Eukaryote - prokaryote ........................................................... 17
RBS and IRES ................................................................................................................................................ 18
Which reporter gene construct can be used? ............................................................................................. 18
Protein purification – HIS-tag ...................................................................................................................... 19
Pure protein – cleaving mechanism HIS-tag ................................................................................................ 19
Notes ........................................................................................................................................................... 20
Les 2 ............................................................................................................................................................... 22
Restriction Enzymes and Nucleic Acid Separation ....................................................................................... 22
Plasmids as Cloning Vectors ........................................................................................................................ 25
Hosts for Cloning Vectors ............................................................................................................................ 26
, Shuttle Vectors and Expression Vectors ...................................................................................................... 26
Les 1
Je kan de werking van verschillende reagentia en chemicaliën die in isolatieprotocollen
worden gebruikt voor plasmide, RNA en genomisch DNA beschrijven.
Lysate
Chemical Function
SDS (anionic detergent; sodium dodecyl Denatures proteins by binding to internal
sulphate) hydrophobic cores
B-mercaptoethanol/ DTT Wreaking disulphide bonds
β-Mercaptoethanol reduces disulphide
linkage of protein, thus denaturing it
Proteinase K Catalyzes the hydrolizes peptide bonds
RNAse A Catalyzes the cleavage of P-O5 bonds in RNA
To remove the contamination of RNA for
DNA purification
Tris-HCI Maintain stable pH
EDTA Chelating agent that binds to calcium (inhibit
enzymes)
Mg2+ and Ca2+ which is present in the
enzymes and reduces the enzyme activity of
DNase and RNase
DNase enzyme requires Mg2+
Phenol (neutral pH, non-polar solution) Denatures protein and removes
phospholipids
remove proteins and polysaccharide
contaminants.
Chloroform Is a nonpolar (hydrophobic) solvent, in which
nonpolar proteins and lipids get dissolved
Chloroform ensures phase separation of the
two liquids because it has a higher density
,gDNA
Cell membrane lysis/enzyme inactivation/RNA degradation
Detergent:
• SDS
Chaotropic agents:
Chemical • Guanidine hydrochloride/iso-
thiocyanate
• Phenol
• B-mercaptoethanol/DTT
• EDTA
Enzymatic • Proteinase K
• RNase A
Protein/lipid/cell debris removal
Organic solvent Phenol/chloroform/isoamyl alcohol
Salt • Sodium chloride
(neutralize the charges on the sugar • Sodium acetate
phosphate backbone)
DNA binding • Silica membrane
• Magnetic beads
DNA precipitation
Alcohol • Ethanol
• Isopropanol (is used to precipitate
the DNA out of the extraction
solution, so we can wash all those
salts and chemicals away and then
dissolve it in our final solvent)
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