Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Bio-protocol (2017-07-01)

Martino Bardelli, Francisco Zarate-Perez, Leticia Agundez, Nelly Jolinon, R Michael Linden, Carlos R Escalante, Els Henckaerts

PMID28660237

ZUSAMMENFASSUNG

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

MATERIALIEN

Produktnummer

Marke

Produktbeschreibung

L3771

Sigma-Aldrich

Natriumdodecylsulfat, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)

77617

Sigma-Aldrich

Phenol–Chloroform–Isoamylalkohol-Mischung, BioUltra, for molecular biology, 25:24:1

W302600

Sigma-Aldrich

Natriumcitrat-Dihydrat, ≥99%, FG

S3264

Sigma-Aldrich

Natriumphosphat, for molecular biology, ≥98.5% (titration)

E9884

Sigma-Aldrich

Ethylendiamintetraessigsäure, ACS reagent, 99.4-100.6%, powder