Materials and Reagents

1. Sera of patients with chronic hepatitis B (100~200 μl)
   
2. QIAamp MinElute virus spin kit (QIAGEN, catalog number: 57704 )
   
3. Ex taq polymerase (Takara Bio Company, catalog number: RR001A )
   
4. Forward primer (XhoI site is underlined): 5’-AAT CTT CTC GAG GAC TGG GGA CCC TGC ACC-3’
   
5. Reverse primer (NcoI site is underlined): 5’-GAG CAG CCA TGG GAA GGA GGT GTA TTT CCG-3’
   
6. Gel/PCR DNA Extraction kit (Real Biotech Corporation, catalog number: YDF100 )
   
7. pGEM-T Easy vector (Promega Corporation, catalog number: A1360 )
   
8. T4 ligase (Takara Bio Company, catalog number: 2011A )
   
9. LB broth high salt (Duchefa Biochemie, catalog number: L1704 )
   
10. MacConkey agar (BD, catalog number: 212123 )
    
11. LaboPass Plasmid Mini Purification Kit (COSMO Genetech, catalog number: CMP0111 )
    
12. HBV WT complete sequence (genotype C, GQ872210)
    
13. Xho I (New England BioLabs, catalog number: R0146S )
    
14. Nco I (New England BioLabs, catalog number: R0193S )
    
15. CutSmart™ buffer (New England BioLabs, catalog number: B7204S )
    
16. HBV 1.2 replicon (Figure 1)
    
17. TE buffer (see Recipes)
    
    
    Figure 1. A plasmid map of the HBV 1.2-mer replicon with XhoI and NcoI sites highlighted
    

Equipment

1. Centrifuge (Eppendorf, catalog number: 5415R )
   
2. Pipet Aid XP (Drummond Scientific Company, catalog number: 4-000-202-E )
   
3. MJ Mini 48-Well Personal Thermal Cycler (Bio-Rad Laboratories, catalog number: PTC-1148 )
   
4. Nanodrop Spectrophotometer (Nanodrop, catalog number: ND-1000 )
   
5. Heat block
   

Procedure