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Samenvatting lab biotechnologie
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Samenvatting van 3 pagina's voor het vak Lab biotechnologie aan de EhB (volledig)
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Lab biotech1. Isolatie van DNA
1.1. Isolatie van DNA uit wangepitheelcellen
- DNA w geïsoleerd met commerciële Chargeswitch kit (magnetische isolatie)
o Cellyse
o DNA / RNA bindt aan magnetische bolletjes
o DNA w opgelost
- Er w altijd op ijs gewerkt
o DNA & RNA breekt af indien niet koel bewaard
- Invloed op de DNA opbrengst
o Gebruikte techniek
o Donor: # cellen die w uitgescheden = versch per persoon
o Type wattenstaafje
- Staal moet onmiddellijk w gebruikt of w bewaard op 4°C (max 2 weken)
o Wangepitheel bevat bact & nucleasen die DNA aantasten
1.2. Concentratiebepaling van DNA mbv een Nanospec
- Nanospec = spectrofotometer
o Meet de concentratie v DNA, RNA & proteïnen (kleine volumes) adhv
lichtabsorptie
- A260/A280 ratio: zuiverheid v DNA aantonen
o Zuiver staal: 1,8 – 2,0
o Hoe hoger de contaminatie, hoe lager A260/A280 ratio
2. Visualisatie van DNA: gelelektroforese
- DNA / RNA mol w gescheiden obv hun lengte
- Migratie nr anode (kathode -> anode)
- Gel = moleculaire zeef
o Kleine mol geraken verder dan grote mol
- Agarose
o Lineair
o Galactaan (lost op in water)
o Afkomstig uit algen
- TBE buffer
o Tris = organische base
o Boorzuur = brengt pH op 0,8
o EDTA = neutraliseert divalente ionen (Ca+, Mg+)
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