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Plant Gene and Trait, 2013, Vol.4, No.6, 30
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Figure 1 Agrose gel electrophoresis of total RNA extracted
from
Miscanthus sinensis
RNA, with high purity and high integrity, was suitable
for the next step for reverse transcription and RT-PCR
amplification.
1.2 RT-PCR amplification of the target genes
With the synthesized cDNA first strand as template
and the two pairs of speicific PCR primers, two targed
PCR products (Figure 2), named as
MsC4H
and
MsCAD
,
were successfully amplified, respectively.
1.3 Characterization of the positive clones of the
target genes
After the two targets PCR products were recovered
from Agarose gel, they were ligated to pMD18-T and
the ligated products were transformed into the
competent cells of
E. coli
DH5α. The single clones of
transformants were cultivated in LB liquid medium
overnigh and then the plasmid werer isolated from
E.
coli
for PCR identification. The PCR products were
identical to the RT-PCR amplification (Figure 3),
which indicated that they were positive recombinants.
Figure 2 Agarose gel electrophoresis of RT-PCR amplification products
Note: M: Molecular Weight Marker; A:
MsC4H
; B:
MsCAD
Figure 3 PCR identification of positive clones