5. For this, total RNA from the transformants is extracted by
using TRIzol reagent according to the supplier’s instructions.
Thereafter, the RNA is used for cDNA synthesis by using
random hexamer primer following the supplier’s specifications.
Subsequently, eGFP and Sh-ble genes are PCR amplified from
the cDNA template to assess the transcription of the introduced genes in the transformants.
4 Notes
1. Cells should be in exponential phase for the preparation of
electrocompetent cells, otherwise the transformation efficiency
would be reduced.
2. The solid agar medium and the antibiotic stock are prepared
freshly.
Fig. 1 Molecular evaluation of electroporated transformants. (a) Putative transformants grown in the IMK agar
plate supplemented with Zeocin result from the successful electroporation mediated transformation of
recombinant plasmid (right), whereas no growth is observed in the selection media under the control
conditions where the cells are electroporated with salmon sperm DNA alone. (b) Subsequent molecular
characterization to evaluate the transformation by semiquantitative RT-PCR. Lane eGFP, amplicons denote the
transcription of eGFP reporter gene in the transformants. Lane Zeocin, amplicon represents the transcription of
Bh-sle marker gene into the host genome which confers Zeocin resistance to the transformants. Lane M, DNA
ladder
178
Da-Wei Li et al.
using TRIzol reagent according to the supplier’s instructions.
Thereafter, the RNA is used for cDNA synthesis by using
random hexamer primer following the supplier’s specifications.
Subsequently, eGFP and Sh-ble genes are PCR amplified from
the cDNA template to assess the transcription of the introduced genes in the transformants.
4 Notes
1. Cells should be in exponential phase for the preparation of
electrocompetent cells, otherwise the transformation efficiency
would be reduced.
2. The solid agar medium and the antibiotic stock are prepared
freshly.
Fig. 1 Molecular evaluation of electroporated transformants. (a) Putative transformants grown in the IMK agar
plate supplemented with Zeocin result from the successful electroporation mediated transformation of
recombinant plasmid (right), whereas no growth is observed in the selection media under the control
conditions where the cells are electroporated with salmon sperm DNA alone. (b) Subsequent molecular
characterization to evaluate the transformation by semiquantitative RT-PCR. Lane eGFP, amplicons denote the
transcription of eGFP reporter gene in the transformants. Lane Zeocin, amplicon represents the transcription of
Bh-sle marker gene into the host genome which confers Zeocin resistance to the transformants. Lane M, DNA
ladder
178
Da-Wei Li et al.