Genomics and Applied Biology 2016, Vol.7, No.5, 1-8
4
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.
Electrophoresis
:
PCR product was submitted to electrophoresis using 1% agarose gel, stained by ethidium
bromide (EB) and visualized under ultraviolet light (UV Trans illuminator).
3 Results
The HBV Creg DNA fragment was isolated by recombinant PCR.
The HBV Creg DNA fragment was amplified from extracted DNA of hepatitis B infected patient serum by using
recombinant PCR. The PCR product was sequenced and then tested by the software chromas based on GenBank
accession number ID: 5536.
Figure 2 (A) and Figure 2 (B) illustrated the recombinant PCR product at 1 401 bp parallel with the DNA size
marker (DNA marker D2000). The PCR product was cloned into the pcDNA3.1 (+) expression vector and named
it pcDNA3.1 (+) HBV Creg, then the recombinant plasmid was transformed in E. coli DH5αstrain, and the
transfected into HepG2 cells.
(A)
(B)
Figure 2 the recombinant PCR result of HBV Creg DNA
Note: (A) electrophoresis of products after 1
st
PCR in 1% agarose gel by Ethidium bromide-staining. 701bp and 731bp products were
amplified using primer P1F/P1R in lane 1 and primer P2F/P2R in lane 2. (B) electrophoresis of products after 3
rd
PCR in 1% agarose
gel. 1401bp product was amplified by recombinant 701bp and 731bp products of 1
st
PCR using primer P1F/P2R, this DNA fragment
was that we wanted
Recombinant plasmid pcDNA3.1 (+) HBV Creg was constructed.
The recombinant plasmid pcDNA3.1 (+) HBV Creg was extracted from E. coli DH5αstrain, and then detected by
PCR and double digested respectively. Figure 3 (A) illustrated the detection result of the recombinant plasmid
pcDNA3.1 (+) HBV Creg by PCR, and Figure 3 (B) illustrated the detection result of the recombinant plasmid
pcDNA3.1 (+) HBV Creg by double digested using blunt end cutter EcoR
Ⅰ
and Nde
Ⅰ
restriction enzyme, both
Figure 3 (A) and Figure 3 (B) illustrated a DNA fragment at 1 401 bp, that indicated the recombinant plasmid
pcDNA3.1 (+) HBV Creg was successfully constructed.
