Pgem t easy plasmid vector

PCR was conducted using the high-fidelity Phusion DNA polymerase (Thermo-Fisher Scientific, Beijing, China) under the following conditions: 2 min of denaturation at 98°C; 35 cycles of 15 s at 98°C, 30 s at 61°C, and 30 s at 72°C; followed by a final extension of 5 min at 72°C. The PCR products were cloned into the pGEM-T Easy Vector plasmid (Promega Corporation, Madison, USA). The recombinant plasmids were then transformed into Escherichia coli DH5α cells, with the positive clones sequenced commercially (Huada Gene, Shenzheng, China). All primers used in this study are listed in Table S6.
The semi-quantitative RT-PCR experiments in this work were conducted following a previous publication (Zhou et al., 2011 (link)). The amplification of wheat tubulin gene transcripts was used to normalize the cDNA contents of various reverse transcription mixtures before PCR, and to monitor the kinetics of thermo-amplification during PCR. The reproducibility of the transcriptional patterns revealed by semi-quantitative PCR was tested by at least three independent assays.

SSH was performed between paired HCC and non-HCC tissue with the Clontech PCR-Select cDNA Subtraction Kit (Clontech, Mountain View, CA, USA) according to the manufacturer's protocol for HBV-associated HCC patients using two-way subtraction. In the forward subtraction, the codes from HCC were used as the tester, and the codes from paired non-HCC tissues were used as the driver; in the reverse subtraction, the codes from the paired non-HCC tissues were used as the tester, and the codes from HCC were used as the driver. Ten nanograms of the PCR product was cloned into the pGEM-T Easy Vector plasmid (Promega, Madison, WI, USA) and transformed into competent E. coli XL2-Blue cells (Stratagene, Cedar Creek, TX, USA). Two thousand colonies were randomly selected. The plasmids were purified on Qiaprep Spin Columns (Qiagen, Hilden, Germany), and the insert sequences were determined by restriction endonuclease digestion with EcoRI. The final sequence homolog searches were performed using the GenBank (nr) and EST (dbEST) databases employing the BLASTN algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST).

cDNA was generated from RNA extracted from SHP77 cells. The DNA sequence coding NRXN1 was reconstructed by polymerase reaction with the first half and the latter half of the entire coding sequence [46 (link)]. The first half of the sequence was amplified with forward primer 5′-TCC CGC CTT TCC CCT TAC-3′ and reverse primer 5′-GCT GGA ATT ACA GTT AAT CCT GAT AC-3′. The latter half of the sequence was amplified with forward primer 5′-GGA GCA TGT TTA TGA AAA TTC AG-3′ and reverse primer 5′-CAT TCC CTG TCT TCT TTT GTA TG-3′. The full-length sequence was subcloned to the pGEM-T Easy Vector plasmid (Promega, Madison, WI). The plasmid was transformed into E. coli DH-5α Competent Cells (TaKaRa Bio) according to the manufacture’s protocol. The sequences of plasmids from colonies of the competent cells were verified after being extracted from transformed DH-5α using the PureYield™ Plasmid Miniprep System (Promega, Madison, WI). The collected plasmids were extracted from competent cells incubated overnight using the PureYield^™ Plasmid Midiprep System (Promega). Fragments including the NRXN1 sequence were subcloned into pcDNA™3.1/Zeo⁽⁺⁾(Thermo Fisher Scientific). HEK293 cells were transfected with HilyMax Reagent (Dojindo, Kumamoto, Japan) and incubated for 2 days, at which time the cells were examined to confirm the overexpression of the target genes using qRT-PCR and flow cytometry.