Genomics and Applied Biology 2016, Vol.7, No.5, 1-8
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The third PCR reaction
:
The PCR reaction contained 10 μl of the second PCR reaction product, 0.05 mM dNTP,
5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The
PCR reaction use primer p1F and p2R), 2.5 unit of Taq DNA polymerase in 50 μl final volumes.
PCR reaction was carried out within 30 cycles: denaturation at 94
℃
for 30 sec, annealing at 56
℃
for 30 sec, and
elongation at 72
℃
for 90 sec.
Electrophoresis
:
PCR product was submitted to electrophoresis using 1% agarose gel, stained by ethidium
bromide (EB) and visualized under ultraviolet light (UV Trans illuminator).
Cloning
:
pcDNA3.1(+) and HBV Creg DNA fragment was digested by blunt end cutter EcoR
Ⅰ
and Nde
Ⅰ
restriction enzyme, the double digested product of HBV Creg DNA fragment was recovered by DNA purified kit
(Promaga 230262), and the double digested product of pcDNA3.1(+) was submitted to electrophoresis using 0.5%
agarose gel and then the larger DNA fragment was recovered by gel DNA extraction kit (TIANGEN I7804), Then
the two DNA fragment that was purified were ligated together by T4 DNA ligation enzyme and transformed in E.
Coli DH5αstrain, and then selected on LB agar containing 100 μg/ml of ampicillin. The transformant colony was
inoculated into 3 ml LB medium and allowed to grow at 37
℃
in a shaker at 200 rpm overnight. The next day,
recombinant plasmid was extracted by plasmid extraction kit (TIANGEN Cat. No I7723) and digested with EcoR
Ⅰ
and Nde
Ⅰ
enzymes and electrophoresed through 0.8% agarose gel. And also, the recombinant plasmid was
examined by PCR. Gel contained DNA fragment (containing HBV Creg DNA fragment) was seizured by scalpel
under long wave UV (Sambrook and Russell, 2001).
DNA sequencing
:
The colony was sent to TIANGEN corporation (Beijing), so as to submit for sequencing by
dideoxy chain termination method.
Assay of gene integration
:
Briefly, the HepG2 cells was transfected with recombinant plasmid by
Lipofectamine
TM
2 000 (Invitrogen 455 000) and selected in RPMI1640 medium containing 100 μg/ml of G418
(MDBio Inc 813 089). The transfected cells was inoculated into 25 ml RPMI 1 640 medium to grow at 37
℃
5%
CO2 in a incubater for about a month and the medium was changed every 2 days. The total DNA of HepG2 cells
was extracted and detected in the day when the HepG2 cells were transfected 4 days and 30 days, Untransfected
control culture was analyzed in parallel.
The total DNA was extracted from the HepG2 cells that were transfected 4 days, 30 days and Untransfected
control culture, then the HBV Creg DNA fragment, HBV X gene fragment and HBV C gene fragment were
detected by PCR respectively, The PCR reaction can be described as:
The HBV Creg DNA extending PCR reaction
:
The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl
10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR
reaction use primer P1F and P2R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.
PCR reaction was carried out within 30 cycles: denaturation at 94
℃
for 30 sec, annealing at 60
℃
for 30 sec, and
elongation at 72
℃
for 40 sec.
The HBV X DNA extending PCR reaction
:
The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl
10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR
reaction use primer P1F and P1R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.
PCR reaction was carried out within 30 cycles: denaturation at 94
℃
for 30 sec, annealing at 60
℃
for 30 sec, and
elongation at 72
℃
for 40 sec.
The HBV C DNA extending PCR reaction
:
The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl
10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR
reaction use primer P2F and P2R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.
