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Te wskazówki dotyczące rozwiązywania problemów mają zastosowanie do większości dostępnych na rynku zestawów do oczyszczania plazmidowego DNA.","table":"

Problem	Możliwa przyczyna	Rozwiązanie
Wydajność plazmidowego DNA jest niska	Kultura bakteryjna nie była świeża.	Kultura powinna być przetwarzana w odpowiednim czasie po osiągnięciu wymaganej gęstości komórek. Alternatywnie, osad bakteryjny może być przechowywany w temperaturze -20 °C przed ekstrakcją plazmidowego DNA bez znaczącego wpływu na czystość lub jakość.
	Plazmid ma niską liczbę kopii.	Przetwórz większą objętość kultury lub hoduj w bogatszym podłożu. Alternatywnie, sklonuj sekwencję DNA będącą przedmiotem zainteresowania do plazmidu posiadającego wyższą liczbę kopii, na przykład pCORON1000 (~300-500 kopii na komórkę).
	System oczyszczania został przeciążony.	Pomiar A₆₀₀ hodowli przed przetwarzaniem, aby upewnić się, że kolumna oczyszczająca nie jest przeciążona resztkami bakterii (np, przy użyciu systemu illustra™ plasmidPrep Mini Spin. Jeśli A₆₀₀ wynosi > 5, zmniejsz objętość przetwarzanej kultury).
	Komórki nie zostały odpowiednio zawieszone.	Resuspensję komórek można osiągnąć poprzez worteksowanie, pipetowanie w górę / w dół lub przesuwanie probówki mikrowirówki zawierającej komórki bakteryjne wzdłuż pokrywy pustego pojemnika na końcówki pipety.
	Komórki nie zostały odpowiednio zlizowane.	Po dodaniu alkalicznego roztworu lizującego należy delikatnie, ale całkowicie wymieszać.
	Szczepy EndA+ (np, HB101) posiadają nukleazy i nadmiar węglowodanów.	W przypadku stosowania formatów oczyszczania opartych na krzemionce, należy zastosować dodatkowe płukanie kolumny oczyszczania plazmidu (po związaniu plazmidowego DNA) przy użyciu buforu o wysokiej zawartości soli. Ten dodatkowy etap usuwa pozostałości nukleaz i nadmiar węglowodanów, zwiększając w ten sposób wydajność i jakość plazmidowego DNA. Alternatywnie, można użyć szczepu EndA^-, takiego jak TOP10 lub DH5α.
Plazmidowe DNA pojawia się jako rozmaz podczas oglądania na żelu agarozowym	Szczepy EndA⁺ (np, HB101) posiadają nukleazy.	W przypadku stosowania formatów oczyszczania opartych na krzemionce, należy zastosować dodatkowe płukanie kolumny oczyszczania plazmidu (po związaniu plazmidowego DNA) przy użyciu buforu o wysokiej zawartości soli. Ten dodatkowy etap usuwa resztkową aktywność nukleazy, zwiększając w ten sposób wydajność i jakość plazmidowego DNA. Alternatywnie, należy użyć szczepu EndA^-, takiego jak TOP10 lub DH5α.
Plazmidowe DNA jest zanieczyszczone genomowym DNA	Liza alkaliczna: Próbka była mieszana zbyt energicznie po dodaniu buforów lizujących.	Mieszaj delikatnie, odwracając próbkę 10-15 razy po dodaniu któregokolwiek z roztworów. Energiczne mieszanie może spowodować ścinanie genomowego DNA, ułatwiając w ten sposób jego kopuryfikację z plazmidowym DNA.
Plazmidowe DNA jest zanieczyszczone RNA	Próbka nie została poddana działaniu RNazy.	RNaza (100 μg/mL) jest zawarta w buforze lizującym zestawu illustra™ plasmidPrep Mini Spin Kit. W przypadku korzystania z innych metod, należy dodać etap RNazy, jeśli nie jest on jeszcze uwzględniony.
Plazmidowe DNA nie tnie się do końca w temperaturze 37 °C	Plazmidowe DNA zostało nieodwracalnie zdenaturowane i dlatego nie będzie cięte.	Delikatnie wymieszaj, odwracając próbkę bakterii 10-15 razy po dodaniu alkalicznego roztworu lizującego. Unikaj przedłużonej ekspozycji (> 5 min) lub NaOH w roztworze do lizy nieodwracalnie zdenaturuje plazmidowe DNA.
	Oczyszczanie na bazie krzemionki: Wszystkie ślady etanolowego buforu płuczącego nie zostały usunięte.	Obecność pozostałości etanolu może wpływać na dalsze zastosowania, dlatego należy go ostrożnie usunąć, na przykład po przemyciu i wysuszeniu kolumny oczyszczającej przez odwirowanie. Jeśli jakikolwiek etanolowy bufor płuczący wejdzie w kontakt z kolumną oczyszczającą po odwirowaniu, należy odrzucić przepływ i ponownie odwirować.

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