Presence and size of amplicons were checked by agarose (1%) gel electrophoresis of 5 μL aliquots of the PCR products. PCR products were cloned directly using the pGEM-T Easy cloning vector kit, following the instructions of the manufacturer (Promega Benelux BV, Leiden, the Netherlands). Plasmid-DNA of clones was isolated from randomly selected clones per library using a GeneJET Plasmid Miniprep Kit (Fermentas GMBH, St. Leon-Rot, Germany). Clones were checked for inserts of the expected size by agarose (1%) gel electrophoresis after EcoRI digestion (5 U, Eco RI- buffer for 3 h at 37°C). Sequencing was performed at the DNA Diagnostics Center of Nijmegen University Medical Center, using the M13 forward primer.
Pgem t easy vector cloning kit
Manufactured by Promega
Sourced in United States
The pGEM®-T Easy Vector Cloning Kit is a convenient tool for the efficient cloning of PCR products. The kit provides a linearized vector with a single 3' terminal thymidine (T) at both ends to improve the efficiency of ligation of PCR products by preventing recircularization of the vector and providing compatible overhangs for PCR products generated by thermostable polymerases. The vector also includes multiple cloning sites for insert identification and downstream applications.
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Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Sanger s method, by Macrogen (1 mentions)
Jm109 escherichia coli cells, by Promega (1 mentions)
Chromo4 real time pcr machine, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Wizard plus sv miniprep, by Promega (1 mentions)
Pureyield plasmid miniprep system, by Promega (1 mentions)
Dna purification system kit, by Promega (1 mentions)
7 protocols using pgem t easy vector cloning kit
1
PCR Amplicon Cloning and Sequencing
2
Cloning and Sequencing of Amplicons
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For each isolate, amplicons scattered in the 450–700-bp region were gel-purified, ligated into the pGEM-T Easy Cloning Vector Kit (Promega, Madison, WI, USA), and transformed into Escherichia coli DH5α competent cells according to the manufacturer’s instructions. Twenty to forty colonies per each cloned amplicon were selected and sequenced using Sanger’s method (Macrogen Inc., Daejeon, Korea). The obtained sequences were aligned with global sequences from GenBank and analyzed using CLC Main Workbench 6.
3
Microbial Diversity Analysis via coxL Genes
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One coxL gene library was derived from each sampling station, resulting in nine fully replicated libraries. Partial coxL gene sequences were PCR-amplified using the universal-coxL assay and cloned in pGEM-T® Easy Vector cloning Kit (Promega, WI, USA). Recombinant colonies were selected, plasmid DNA extracted following standard procedure (Sambrook and Russell, 2001 ) and coxL inserts were PCR-amplified and sequenced using the Sanger's Method (Génome Québec Innovation Centre, McGill University, QC, Canada). In total, 279 clones were obtained. Clone sequences were aligned and in silico translated to verify the canonical signature of the active site characterizing type I and type II coxL sequences. The OTU representative sequences (0.90 similarity cut-off) obtained using the universal-coxL assay were deposited in the GenBank database with accession numbers KJ395119 to KJ395310. UniFrac distance matrix, reflecting the pairwise phylogenetic distance between the sequences retrieved from each sampling site was calculated to verify if land-use types have significantly different microbial communities (Lozupone and Knight, 2005 (link)).
4
Microbial Diversity in Leech Shed Mucus
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To assess the microbial diversity within leech shed mucus, total DNA was extracted from 3 d old mucosal sheds of two individuals, using the Holmes-Bonner protocol (Holmes and Bonner, 1973 (link)), and subjected to PCR using 27F′ and 1492R′ general eubacteria primers (Lane, 1990 ; Weisburg et al., 1991 (link)) (Ta [annealing temperature] = 50°C; 28 cycles; amplicon ~1450 bp). PCR products were cloned using the pGEM-T Easy Vector cloning kit (Promega, WI), with subsequent transformation into JM109 Escherichia coli cells (Promega, WI). Inserts were amplified using M13F′ and M13R′ vector primers (Ta = 46°C; 35 cycles; amplicon ~1636 bp) and digested with HaeIII restriction endonuclease (NEB, Ipswich, MA, USA) for RFLP typing. Clones with unique restriction profiles were purified and subject to Sanger sequencing using M13 primers with an ABI Genetic Analyzer 3130xl at the WVU Department of Biology Genomics Center. The DNA sequences were aligned and assembled into contigs and identified to the highest taxonomic level possible using nucleotide Basic Local Alignment Search Tool (BLASTn, http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
5
Quantitative Analysis of Rhizosphere Bacteria
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Quantitative real time PCR (q-PCR) was performed on a Chromo4 real time PCR machine (Bio-Rad) to measure the presence and concentration of bacterial 16S rRNA gene associated with the rhizosphere fractions. The reactions were performed with IQ SYBR Green Supermix (Bio-Rad), using primers targeting the 16S rRNA gene (Bac357-F and Bac907-R) [16 (link)]. PCR SYBR green reactions were prepared by using the “Brilliant SYBR Green QPCR Master Mix” kit (Stratagene) in 96-well plates. The reaction mix (25 mL) contained 1X Brilliant SYBR Green (2.5 mM MgCl2), 0.12 mM of each primers, and approximately 100 ng of extracted DNsA. The DNA obtained from the three plants sampled in the same station was pooled and used as template to carry out the real time assay in triplicate. At the end of each real time PCR, a melting curve analysis was performed for verifying the specificity of PCR products. To construct standard curves, the 16S rRNA gene of Asaia sp. was amplified by PCR and cloned using the pGEM T-easy Vector Cloning Kit (Promega). q-PCR data relative to the 16S rRNA gene concentration were log-transformed.
6
Cloning and Sequencing of DGGE Bands
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Selected bands were excised from DGGE gels with a sterile razor, placed in 40 μL sterile water and incubated at 4 °C for diffusion of DNA into the water. The bands were chosen based on their appearance intensities in DGGE. The DNA was cloned into pGEM®-T Easy Vector Cloning Kit (Promega) according to the manufacturer’s protocol. Competent JM109 E. coli cells were transformed and plated. Plasmid DNA was purified with Wizard® Plus SV Minipreps (Promega) and quantified with Nanodrop (Thermoscientific, Waltham, MA, USA) from colonies containing inserts. The DNA samples were then sent for sequencing to AGRF (Australian Genome Research Facility) according to AGRF requirements (www.agrf.org.au ). The sequences obtained were compared to available database sequences for bacteria using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/ ). Sequences with identity >95% were considered to represent the same taxonomic group.
7
Cloning and Sequencing of PCR Products
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To purify the PCR products, 40 μl of each PCR reaction mix was loaded on a 1 % low melting point agarose gel. The specific amplified fragments bands (800-1000bp in ITS and 600-1500 bp in 28s, IGS and ETS) were cut out and purified with Promega DNA purification system kit. PGEM-T Easy Vector cloning kit (Promega) was then used following the manufacturer’s instructions to clone the purified PCR products. The vectors are prepared by cutting the pGEM®-T Easy Vectors, with EcoRI and adding a 3′ terminal thymidine to both ends. Insertional inactivation of the alpha-peptide allows recombinant clones to be directly identified by blue/white screening on indicator plates. PureYield™ Plasmid Miniprep System (Promega) was used for plasmid purification before being sent for sequencing (GATC-biotech, Germany).
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