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B05MBT Samenvatting Moleculaire Biologie
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B05MBT Samenvatting Moleculaire Biologie Theorie. Ik heb een 8,6 gehaald voor het tentamen, door deze samenvatting.
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Moleculaire BiologieTheorie
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1. De PCR................................................................................................................................................5
1.1. Werking van de PCR.....................................................................................................................7
1.2. PCR protocol................................................................................................................................8
1.3. Wat heb je nodig bij een PCR?.....................................................................................................8
1.3.1. Template DNA.......................................................................................................................9
1.3.2. Overige ingrediënten in de PCR-reactie................................................................................9
1.4. Polymerases en proofreading....................................................................................................11
2. Ontwerpen van primers....................................................................................................................12
2.1. Algemene regels voor primer design.........................................................................................14
3. Kloneren...........................................................................................................................................15
3.1. Plasmiden..................................................................................................................................15
3.2. Stap 1: Gebruik van restrictie-enzymen om DNA te knippen en plasmide te lineariseren........17
3.2.1. Type II endonucleases geven sticky-ends of Blunt ends.....................................................18
3.2.2. Relaties tussen restrictie-enzymen.....................................................................................18
3.2.3. Knippen met 1 of 2 RE.........................................................................................................19
3.3. Stap 2: Ligeer een DNA fragment in een plasmide.....................................................................19
3.3.1. Ligase protocol....................................................................................................................19
3.4. Primers ontwerpen met restrictie-sites.....................................................................................20
3.5. Digestie controle (insert oriëntatie)...........................................................................................21
3.6. Controle van gemaakt plasmide................................................................................................22
3.7. Stap 3: transformatie in bacteriën.............................................................................................23
3.8. Stap 4: identificeer en isoleer een kolonie met het target DNA................................................24
4. Transformeren en Transfecteren......................................................................................................25
4.1. Transformeren van eukaryote cellen; injecteren, transfecteren en transductie.......................25
5. Isoleren van DNA, RNA en eiwitten..................................................................................................27
5.1. Orgaan, weefsel of biopt homogeniseren..................................................................................28
5.2. Lyseren of sonificeren van cellen, micro-organismen en kleine weefsels..................................29
5.3. Scheiden van bestandsdelen dmv centrifugatie........................................................................32
5.4. Isoleren van nucleinezuren (RNA en DNA).................................................................................33
5.4.1. DNA/ RNA hoeveelheid & kwaliteit....................................................................................34
6. De Real-Time qPCR...........................................................................................................................35
6.1. RNA............................................................................................................................................35
6.1.1. Werken met RNA................................................................................................................35
6.2. Meten van RNA met een Northern blot.....................................................................................36
, 6.3. Meten van RNA met de real-time qPCR.....................................................................................37
6.3.1. Van RNA naar cDNA............................................................................................................37
6.3.2. Hoe meet je tijdens een RT qPCR?......................................................................................38
6.4. Detectie, analyse en kwantificering...........................................................................................40
6.4.1. Efficiëntie van de qPCR.......................................................................................................41
6.4.2. Het kwantificeren van de qPCR-data..................................................................................42
7. Sequencing.......................................................................................................................................43
7.1. Sanger sequencing.....................................................................................................................44
7.2. 2nd generation sequencing.......................................................................................................45
7.2.1. Illumina sequencing............................................................................................................45
7.2.2. Ion torrent..........................................................................................................................46
7.3. 3rd generation sequencing........................................................................................................46
8. Eiwitsequenties en massa spectrometrie.........................................................................................47
8.1. 5 fasen in massa analyse in een massa spectrometer...............................................................47
8.2. MALDI-TOF.................................................................................................................................48
8.3. Elektrosprayionisatie (ESI).........................................................................................................48
8.4. Tandem mass spectrometry......................................................................................................49
8.5. Eiwit analyse..............................................................................................................................50
9. Enzymkinetiek...................................................................................................................................51
9.1 Enzymen.....................................................................................................................................51
9.2. Remming van enzymen..............................................................................................................54
9.3. Enzymkinetiek............................................................................................................................55
9.3.1. Michaelis-Menten verzadigingscurve.................................................................................56
9.3.2. IC50.....................................................................................................................................58
10. Eiwitisolatie/zuivering....................................................................................................................59
10.1. Uitzouten.................................................................................................................................60
10.1.1. Ontzouten.........................................................................................................................61
10.2.1. Ionwisselingschromatografie (IEC)....................................................................................63
10.2.2. Hydrofobe interactie chromatografie...............................................................................64
10.2.3. Affiniteitschromatografie..................................................................................................65
10.2.4. Gelfiltratie/ gelpermeatie chromatografie........................................................................66
10.3. Volgorde van verschillende methoden....................................................................................67
11. Eiwit analyse...................................................................................................................................68
11.1. SDS-PAGE.................................................................................................................................68
11.2. Iso-elektrisch focussing............................................................................................................69
11.3. 2D elektroforese......................................................................................................................69
, 11.4. Western blot............................................................................................................................69
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