Quantification of HBV cccDNA via Plasmid-Safe Digestion

Total cellular DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and 200 ng of extracted DNA was subjected to plasmid-safe digestion (Epicenter, WI, USA) in a total reaction volume of 20 μl. The extraction procedure, with DNA from HBV transgenic mice as control for elimination of non-cccDNA, has been described previously^{7 (link), 24 (link)}. Digestions were carried out for 16 hours at 37 °C and the enzyme was inactivated following incubation at 70 °C for 30 min. Five microliters of treated DNA was amplified using FastStart Universal SYBR Master (Roche, Basel, Switzerland) with S primers (Supp. Table 4) and HBV DNA quantified as described earlier.

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Scott T., Moyo B., Nicholson S., Maepa M.B., Watashi K., Ely A., Weinberg M.S, & Arbuthnot P. (2017). ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells. Scientific Reports, 7, 7401.

Publication 2017

QIAamp DNA Blood Mini Kit FastStart Universal SYBR Master

Blood Cellular Digestions Enzyme Plasmid Primers Transgenic mice

Corresponding Organization :

Other organizations : University of the Witwatersrand, South African Medical Research Council, National Institute of Infectious Diseases

Top 5 similar protocols

Variable analysis

independent variables

Plasmid-safe digestion

dependent variables

HBV DNA quantity

control variables

Total cellular DNA extracted using QIAamp DNA Blood Mini Kit
DNA from HBV transgenic mice for elimination of non-cccDNA

controls

Positive control: DNA from HBV transgenic mice
Negative control: Not explicitly mentioned

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