Pmd18 t simple vector

Hemocyte total RNA was extracted as described above, genomic DNA was digested using RNase-free DNase (Promega), and first-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega). The cathepsin L cDNA was amplified using ExTaq DNA polymerase (Takara) and PCR amplification in this case was performed over 32 cycles at 95°C for 5 min, at 54°C for 30 s, and at 72°C for 1 min. The PCR product was gel-purified, cloned into a pMD18-T simple vector (Takara), and verified by sequencing. The cathepsin L cDNA was further amplified using primers with endonuclease sites, which are summarized in Table 1.

Based on the result of de novo transcriptome sequencing of R. irregularis colonized L. barbarum roots, two novel full-length cDNA sequences encoding potassium channel protein were obtained, and designated LbKT1 and LbSKOR. Subsequent PCR was performed using cDNA from L. barbarum roots (both AM and NM treatment under WW condition) as template. Purified PCR products were transformed into the pMD18-T simple vector (Takara) for sequencing (Sunnybio, www.sunnybio.cn).
The sequence data were subject to similarity analysis using the BLAST program in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The DNASTAR program was used to analyze the open reading frame (ORF), and to predict the amino acid sequence. Signal peptide analysis was performed using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). Subcellular localization was predicted using PSORT Prediction (http://psort.hgc.jp/form.html). Transmembrane protein structure was predicted by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) and TMHMM server v2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Conservative domain was predicted by BLASTP in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA 5.05 (Tamura et al., 2011 (link)).

The process for Dzungharian basin oilfield soil layer 16S rRNA gene clone library construction, RFLP analysis, and sequencing of the representative clones were conducted mainly according to the description provided by Liang et al. [17 (link)]. Briefly, the 16S rRNA genes of the soil bacteria were amplified using 27F/1492R as the primer and soil DNA as the template. PCR products were then cloned into pMD18-T simple vector (Takara, Dalian, China) to construct the clone libraries. For each soil layer, two clone libraries were constructed. The RFLP analysis was conducted using Hha I and Msp I to digest the plasmid carrying the 16S rRNA gene, and the fingerprint of every clone was recorded. The fingerprints were then converted into a two-dimensional binary matrix through a binary scoring system (1 for the presence of a band and 0 for the absence). Based on this binary matrix, the similarity between the clone and the OTU was determined (Applied Biostatistic, Setauket, NY). A representative clone of every OTU was selected for 16S rRNA gene sequencing, and the sequences obtained from this study were deposited in GenBank under accession numbers KT353550-KT353563 and KT893461-KT893475. The 16S rRNA gene sequences of clones were aligned, and the phylogenetic analysis were carried out using the PHYLIP package (http://www.phylip.com/).

Xie et al.13 (link). reported that an EST sequence of HOT is over-expressed in the THR strain of B. tabaci. The 3′ and 5′ RACE reactions were constructed using the SMART^TM RACE amplification kit (Clontech, USA) according to the manufacturer’s protocol. All primers used are listed in Table 2. The PCR products were cloned into the pMD18-T simple vector (TaKaRa, Japan). At least 10 clones from both the 5′- and 3′-RACE libraries were sequenced using both M13 forward and M13 reverse primers.

The human HOXA11 open reading frame (ORF) was PCR-amplified by using HOXA11 cDNA as template. The amplification generated Bam HI and Xba I sites into the 5′ and 3′ ends of the HOXA11 ORF. The PCR product was inserted into pMD18-T simple vector (Takara, Dalian, Liaoning, P. R. China). Site-directed mutagenesis was performed on this template to generate a HOXA11 sequence harboring the p. E255K mutation. Then the wild-type and mutated HOXA11 ORF were cloned into pcDNA3.1(+) expression vector (Invitrogen Life Technologies, Carlsbad, CA, USA), respectively. A FOXO1 expression plasmid was created by cloning its ORF into the Bam HI and Xba I sites of the pcDNA3.1(+) vector. For the reporter plasmid, the promoter region of the human PRL gene (from −519 to +65) was cloned into the Kpn I and Nhe I sites of pGL3-basic vector (Promega, Madison, WI, USA). All the plasmids were verified by sequencing. The PCR primers used for plasmid construction are shown in Additional file 1: Table S2.

The forward primers CaXMT-F1 and TCS-F1 (Table 1) both contained a starting codon and a BamHI restriction site, while the reverse primers CaXMT-R1 and TCS-R1 (Table 1) contained SalI. PCR reactions were performed using PrimeSTAR HS DNA polymerase (Takara) under the following PCR conditions: Denaturation at 98°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 1 min, repeated for 30 cycles. The PCR products were sub-cloned into pMD18-T Simple vector (Takara). Nucleotide sequencing was carried out to confirm the resulting plasmids (Invitrogen Pasadena, CA, USA). The excised BamHI-SalI fragments of CaXMT1 and TCS1 were cloned into the GST fusion vector pGEX-4t-2 at the corresponding sites (TransGene Biotech, Beijing, China). The resulting plasmids were transformed into the expression host E. coli Rosetta (BL21, DE3) by heat shock. Empty vector was also transformed as a control.

Genomic DNA extracted from the KYSE70 cell line using the Takara MiniBEST Universal Genomic DNA Extraction kit (Takara), was modified by sodium bisulfite using the EpiTect Bisulfite kit (Qiagen, Germany) according to the manufacturer’s instructions. Methylation status was analyzed by bisulfite genomic sequencing (BSP) of the CpG islands. The region was amplified using the primers shown in Table I. Amplified products were cloned into pMD-18T simple vector (Takara), transformed into DH5α competent cells (Takara), and plated under ampicilin selection. Five independent clones were sequenced.