Pcr select cdna subtraction kit

A subtracted cDNA library was constructed by SSH using a PCR-select™ cDNA subtraction kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. A total of 2 μg mRNA was used for each first-strand cDNA synthesis. The cDNA obtained from the HepG2 cells transfected with pcDNA3.1 (−)−NS2 (genotype 1b; Beijing Key Laboratory of Emerging Infectious Diseases, Beijing Ditan Hospital) was used as a tester and the cDNA from the HepG2 cells transfected with pcDNA3.1 (−) was used as a driver. Subsequently, the subtracted PCR products were cloned into pGEM-T Easy vectors (Promega Corp., Madison, WI, USA). The ligation reactions were then transformed into chemically competent DH5α cells (China Infrastructure of Cell Line Resource) using standard molecular biology techniques.
Following sequencing of the positive colonies (Shanghai BioAsia Biotechnology, Shanghai, China), the basic local alignment search tool (BLAST) server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) at the National Center for Biotechnology Information (Bethesda, MD, USA) was used to identify nucleic acid homology.

Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) from samples of the different years, and the contaminating genomic DNA was removed by DNAase I (Qiagen) treatment followed by a clean-up with the RNeasy MinElute Cleanup kit (Qiagen). cDNA was then synthesized from pistil, leaf, and mature pollen total RNA using the SMART PCR cDNA Synthesis kit (Clontech). The subtracted libraries were constructed with the PCR-Select cDNA Subtraction Kit (Clontech). A total of 6 libraries were constructed: 1. Pistil subtracted with pollen [P(Po)]; 2. Pollen subtracted with pistil [Po(P)]; 3. Pistil subtracted with leaf [P(L)]; 4. Leaf subtracted with pistil [L(P)]; 5. Pollen subtracted with leaf [Po(L)]; 6. Leaf subtracted with pollen [L(Po)], according to the manufacturer's instructions. Two rounds of PCR amplifications were also performed according to the manufacturer's protocol in order to enrich differentially regulated genes, by using the PCR Primer 1 and the Nested PCR primer 1 and 2R as indicated in the manufacturer's instructions and provided by the kit.

RNA was extracted with the use of the Trizol (Invitrogen) system following the manufacturer's instructions and RNA samples were submitted to Dnase to remove traces of DNA. The PCR-select cDNA subtraction kit (Clontech) was used in order to obtain differentially expressed transcripts [17 (link)] according to the manufacturer's instructions. Subtractions were carried out from EGG and 452 samples. Fragments of cDNA subtracted were extracted from gel, purified by Wizard SV gel kit and PCR clean-up system (Promega), and then cloned into pGEM-T easy vector (Promega) according to the manufacturer's instructions. Cloning was carried out using Escherichia coli bacteria DH5α (Life Technologies). Individual colonies were grown in 100 μL LB-ampicillin at 37°C, plasmid was isolated, and the presence of the insert was confirmed by digestion reaction using EcoRI (5 U) followed by a PCR using nested primers (Clontech). For those clones presenting insert, plasmidial DNA was isolated from bacterial cultures using Wizard plus SV minipreps (Promega) kit. Positive clones were sequenced in accordance with a method previously described [18 (link)], making use of DYEnamic ET dye terminator kit (MegaBACE, GE Healthcare).

SSH was performed with the PCR-Select™ cDNA subtraction kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. In brief, 2.0 μg of poly A+ mRNA, each from the pcDNA3.1(−)-MHBs^t167 tester group and the pcDNA3.1(−) driver group was subjected to cDNA synthesis, respectively. Following restriction with RsaI, small sizes of cDNAs were obtained. The tester cDNAs were then subdivided into two parts, ligated with the specific adaptor 1 and adaptor 2, respectively. After two subtractive hybridization reactions and two suppression PCR amplifications, differentially expressed cDNAs were selectively amplified. Subsequently, the second PCR products were used as templates for PCR amplification of G3PDH (a housekeeping gene) at 18, 23, 28, 33 cycles, respectively, to analyze subtraction efficiency. The second PCR products were directly purified using the Wizard^® PCR-Preps DNA Purification system (Promega), and inserted into pGEM-T Easy (Promega) to construct the subtracted library. Colony PCRs were conducted to confirm that the size of the cDNA inserts ranged between 200 and 1,000 bp by using T7/SP6 specific primers localized in pGEM-T Easy. Following DNA sequencing of the positive colonies, nucleotide homology searches were performed using the BLAST program at NCBI (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi).

The two cDNA libraries used for generating differentially expressed SSH libraries were synthesized from 250ng of DNase-treated total RNA isolated from FR light-induced haustoria or stems 3 d after light treatment (for each RNA isolation, material was pooled from six induced shoots) using the SMARTer Pico PCR cDNA Synthesis kit (Clontech, Mountain View, CA, USA). SSH was carried out with the PCR-Select cDNA Subtraction Kit (Clontech). The Advantage 2 PCR Kit (Clontech) was used for all PCR amplifications. After amplification, the differentially expressed cDNAs were cloned into the pGEM-T Easy vector system (Promega, Madison, WI, USA). One Shot TOP10 Chemically Competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) were transformed with the SSH libraries and incubated on LB/Carbenicillin/X-Gal/IPTG plates at 37 °C for blue/white screening. All procedures were carried out according to the manufacturers’ instructions. Plasmid DNA was isolated from white colonies by alkaline lysis (Birnboim and Doly, 1979 (link)) and sequenced with M13F primer (5′ GTAAAACGACGGCCAGT 3′) by Sanger sequencing (Macrogen Korea, Seoul, Korea).

Total RNA was extracted from L693 and L661 leaves using the RNA extraction reagent TRIzol (Invitrogen, Carlsbad, CA, USA). The Dynabeads Oligo dT₂₅ system (Dynal A.S., Oslo, Norway) was then employed to purify mRNA and to construct cDNA libraries produced by RT-PCR. An SSH library was constructed using the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA, USA) according to a previously described protocol [30 (link), 35 (link)]. Positive colonies were sequenced using an ABI Prism 3100 automated sequencer (Perkin Elmer ABD, Santa Clara, CA, USA). The resulting ESTs were analyzed against the GenBank database using BLASTX and BLASTN. The threshold probability for a sequence match was set at 10⁻⁵.

Total RNA was extracted using Trizol (DingGuo, China) according to the manufacturer’s instructions and the mRNA was isolated using PolyA Ttract® mRNA Isolation System III (Z5300) Kit (Promega, USA) as described in the user manual. The quality and quantity of total RNA and mRNA were assessed using a 1% sepharose gel.
A “forward” subtractive library was constructed using the PCR-Select™ cDNA subtraction kit (Clontech, USA). The mRNA isolated from HUVECs and HCMV-infected HUVECs (24 h) were designated as “tester” and “driver”, respectively. The final PCR products were purified using PCR Product Recovery kit (DingGuo) and then cloned into the pMD19-T vector (TaKaRa, China), which was then transformed into Escherichia coli DH5a cells. Transformed cells were plated onto standard LB/ampicillin/X-gal/IPTG plates at 37°C for blue/white screening. A certain number of the white colonies were taken for subsequent analysis of the sequence. Colony PCR was employed to amplify the inserted cDNA fragments with pMD19-T vector universal primers M13-47 and M13-48 (M13-47:5-CGCCAGGGTTTTCCCAGTCACGAC-3, M13-48:5-AGCGGATAACAATTTCACACAGGA-3).