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Samenvatting sv geintegreerd practicum voor het theorie examen
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geïntegreerd practicum: moleculaire biologie en geneticaexperiment I: sequentiebepaling van een onbekend gen.....................................................................................1
extractie van DNA met zoutprecipatie..............................................................................................................2
concentratiebepaling van DNA en lambda-faag DNA.......................................................................................2
controle van DNA zuiverheid en concentratie..................................................................................................3
PCR.....................................................................................................................................................................4
transformatie efficientie....................................................................................................................................5
ligatie PCR product in een t-tailed vector.........................................................................................................5
kolonie pcr.........................................................................................................................................................6
Principe plasmide bereiding..............................................................................................................................7
sequentiebepaling.............................................................................................................................................7
Experiment II: Genotypering van polymorfe DNA merkers – Segregatieanalyse.................................................9
genetische merkers...........................................................................................................................................9
gradient PCR......................................................................................................................................................9
paternaliteitsbepaling.......................................................................................................................................9
Analyseren van resultaten TPA 25 en VNTR D1S80........................................................................................11
experiment IIi: SNP of mutatie detectie..............................................................................................................12
Principe HRM en analyseren van resultaten...................................................................................................12
sybr gen...........................................................................................................................................................12
Taqman probes................................................................................................................................................12
smeltcurve.......................................................................................................................................................13
resultaten........................................................................................................................................................14
Experiment IV: Differentiële expressie hAPP in APP23 muismodel vs. controle.................................................16
QPCR................................................................................................................................................................16
Principe van Q-PCR en analyseren van resultaten..........................................................................................16
Experiment V: RPLF-PCR......................................................................................................................................19
Principe van RFLP-PCR.....................................................................................................................................19
EXPERIMENT I: SEQUENTIEBEPALING VAN EEN ONBEKEND GEN
1
, EXTRACTIE VAN DNA MET ZOUTPRECIPATIE
Als we onbekend gen willen detecteren nood aan DNA, we zullen dus DNA isolatie van elke
student uitvoeren
VERSCHILLENDE METHODEN OM GENOMSICH DNA UIT WEEFSEL TE ISOLEREN
§ Fenol-chloroform extractie:
• Veel gebruik
• Producten giftig voor mens en milieu
• Vaak gebruikt bij RNA
§ Extractie mbv zoutprecipitatie
• Veilig
• Gebruiken wij
§ Extractie via DNA isolatie kit met spin kolommen
• Weinig DNA opbrengst
STAPPEN
1. Mond spoelen met 0,8% NaCl om zo wangslijmvlies cellen te isoleren
2. Cellen collecteren via centrifugatie
§ 2 types
• Vaste rotor
• Swinging bucket minder kans hebben dat partikels naar de kant geduwd
worden (minder cellyse) cellen nog niet breken door een langere
sedimentatie afstand
3. Toevoegen lysisbuffer (10 mM Tris pH 8,0, 10mM EDTA, 0,1M NaCl, 2%SDS)
§ Om cellen open te breken daarna verwarmen waardoor de volledige inhoud van
de cel vrijkomt
4. Toevoegen proteïnase K + incubatie
5. Toevoegen verzadigd NaCl en centrifugatie
§ Zal de eiwitmantel rond de eiwitten verbreken en laten neerslaan
6. Supernatans bevat DNA neerslagen met Ethanol
§ DNA is heel negatief geladen dus zal verbonden blijven?
§ Ethanol = (organisch solvent)
§ Kijken (in het midden van het epje zal je zien drijven want DNA is heel viskeus, door
snelle draaiing lucht bellen, als we geen belletjes zien geen DNA of een zeer lage
concentratie) hoe beter opgelost hoe groter de waarde
7. Terug oplossen in TE-4 buffer
CONCENTRATIEBEPALING VAN DNA EN LAMBDA-FAAG DNA
Via spectrofotometrie
2
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