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Tyto tipy pro řešení problémů jsou použitelné pro většinu komerčně dostupných souprav pro purifikaci plazmidové DNA.","table":"

Problém	Možná příčina	Řešení
Výtěžnost plazmidové DNA je nízká	Bakteriální kultura nebyla čerstvá.	Kultura by měla být zpracována včas poté, co dosáhne požadované hustoty buněk. Alternativně lze bakteriální pelety před extrakcí plazmidové DNA skladovat při -20 °C bez významného vlivu na čistotu nebo kvalitu.
	Plazmid má nízký počet kopií.	Zpracujte větší objem kultury nebo pěstujte v bohatším médiu. Případně naklonujte sekvenci DNA, která vás zajímá, do plazmidu s vyšším počtem kopií, například pCORON1000 (~300-500 kopií na buňku).
	Purifikační systém byl přetížen.	Před zpracováním změřte A₆₀₀ kultury, abyste se ujistili, že purifikační kolona není přetížena bakteriálními zbytky (např, při použití systému illustra™ plasmidPrep Mini Spin. Pokud je A₆₀₀ > 5, snižte objem zpracovávané kultury).
	Buňky nebyly dostatečně resuspendovány.	Resuspenze buněk lze dosáhnout buď vířením, pipetováním nahoru/dolů, nebo přejížděním mikrocentrifugační zkumavky obsahující bakteriální buňky po víku prázdného boxu s pipetovací špičkou.
	Buňky nebyly dostatečně lyzovány.	Při přidávání alkalického lyzačního roztoku jemně, ale úplně promíchejte.
	Kmeny EndA+ (např, HB101) mají nukleázy a nadměrné množství sacharidů.	Při použití purifikačních formátů na bázi oxidu křemičitého použijte dodatečné promytí kolonky pro purifikaci plazmidu (po navázání plazmidové DNA) pomocí pufru s vysokým obsahem soli. Tento dodatečný krok odstraní zbytkové nukleázy a nadměrné množství sacharidů, čímž se zvýší výtěžnost a kvalita plazmidové DNA. Případně použijte kmen EndA^-, například TOP10 nebo DH5α.
Plazmidová DNA se při pohledu na agarózový gel jeví jako šmouha	Kmeny EndA⁺ (např, HB101) mají nukleázy.	Při použití purifikačních formátů na bázi oxidu křemičitého použijte dodatečné promytí kolonky pro purifikaci plazmidu (po navázání plazmidové DNA) pomocí pufru s vysokým obsahem soli. Tento dodatečný krok odstraní zbytkovou nukleázovou aktivitu, čímž se zvýší výtěžnost a kvalita plazmidové DNA. Případně použijte kmen EndA^-, například TOP10 nebo DH5α.
Plasmidová DNA je kontaminována genomovou DNA	Alkalická lýza: Vzorek byl po přidání lyzačních pufrů příliš intenzivně promíchán.	Po přidání některého z roztoků vzorek jemně promíchejte 10-15x převrácením. Energické míchání může způsobit střih genomové DNA, a tím usnadnit její kopurifikaci s plazmidovou DNA.
Plazmidová DNA je kontaminována RNA	Vzorek nebyl ošetřen RNázou.	RNáza (100 μg/ml) je součástí lyzačního pufru soupravy illustra™ plasmidPrep Mini Spin Kit. Při použití jiných metod přidejte krok s RNázou, pokud již není obsažena.
Plasmidová DNA se při teplotě 37 °C nedořezává	Plasmidová DNA byla nevratně denaturována, a proto se nedořezává.	Po přidání alkalického lyzačního roztoku bakteriální vzorek jemně promíchejte 10-15x převrácením. Vyhněte se delšímu působení (> 5 min), jinak NaOH v lyzačním roztoku nevratně denaturuje plazmidovou DNA.
	Purifikace na bázi oxidu křemičitého: Všechny stopy ethanolového promývacího pufru nebyly odstraněny.	Přítomnost zbytkového ethanolu může ovlivnit následné aplikace, a proto musí být pečlivě odstraněn, například po promytí a vysušení purifikační kolony centrifugací. Pokud se etanolový promývací pufr dostane po odstředění do kontaktu s purifikační kolonou, zlikvidujte průtok a znovu odstřeďte.

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