Pgem t easy vector system kit

Manufactured by Promega

Sourced in United States, Italy

The pGEM®-T Easy Vector System kit is a cloning vector that provides a convenient system for the cloning and sequencing of PCR amplified DNA fragments. The vector is pre-cut with EcoRI and has a single 3' terminal thymidine residue at each end, which allows for the efficient ligation of PCR products generated by thermostable DNA polymerases that add a single deoxyadenosine to the 3' ends of the amplified fragments.

Automatically generated - may contain errors

Lab products found in correlation

3500 genetic analyser, by Thermo Fisher Scientific (2 mentions) Taqman probe, by Roche (2 mentions) Plasmid miniprep kit, by Promega (2 mentions) Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Pop7 polymer, by Thermo Fisher Scientific (1 mentions) Nanodrop nd 1000 spectrophotometer, by Promega (1 mentions) Genomewalker universal kit, by Takara Bio (1 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Pet28a, by Promega (1 mentions) Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Abi prism 3130 automated sequencer, by Thermo Fisher Scientific (1 mentions) Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions) Agarose gel, by Promega (1 mentions) Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PGEM-T Easy (832 citations) PGEM-T vector (674 citations) PGEM-T (255 citations) T-easy vector (36 citations) T-vector (34 citations) PGEM vector (33 citations) PGEM-T Easy kit (24 citations) PGEM-T easy vector kit (15 citations) PGEM-T kit (9 citations) PGEMs-T Easy Vector System kit (2 citations)

9 protocols using pgem t easy vector system kit

Cloning and Sequencing Cryptosporidium DNA

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cloning technique was used when it was not possible to reliably identify the specimens solely via sequencing. The material studied was cloned using the pGEM^®-T Easy Vector System kit (Promega) following the manufacturer's directions and the method described by Couto et al. (2014) (link). After linking the PCR products from GP60 gene target, the vectors were electroporated and introduced into Escherichia coli cells. Bacteria containing the Cryptosporidium DNA fragment were plated into Petri dishes containing ampicillin (100 μg/ml), X-GAL (80 μg/ml), and IPTG (0.5 mM). The Petri dishes were incubated for 24 h at 37 °C to allow the colonies to grow. Three colonies were randomly selected to be amplified using the primers from the nested-PCR reaction (of the cloned target gene) to ensure that the Cryptosporidium DNA was present. The PCR products were purified using the Wizard Plus System kit (Promega), before being sequenced again.
The same protocol previously described using the 18S and GP60 genes was used for sequencing the cloned samples except for the primers used. Plasmid DNA was sequenced using the universal primers M13F-pUC (GTT TTC CCA GTC ACG AC - Forward) and M13R-pUC (CAG GAA ACA GCT ATG AC - Reverse) as suggested by the manufacturer of the pGEM®-T Easy Vector System kit (Promega).

Mariné Oliveira G.F., do Couto M.C., de Freitas Lima M, & do Bomfim T.C. (2016). Mussels (Perna perna) as bioindicator of environmental contamination by Cryptosporidium species with zoonotic potential. International Journal for Parasitology: Parasites and Wildlife, 5(1), 28-33.

+ Open protocol

+ Expand

Cloning and Sequencing of Bacterial 16S rDNA

Check if the same lab product or an alternative is used in the 5 most similar protocols

The purified PCR products were sequenced and cloned into the PGEM vector following the instructions provided by the pGEM®-T Easy Vector System kit (Promega, Italy) using Escherichia coli competent cells as a host. The obtained plasmids, pGEM-BB (pGEM + B. breve) and pGEM-LS (pGEM + L. salivarius), were extracted (Plasmid Miniprep Kit, Promega, Italy), quantified using the NanoDrop ND-1000 spectrophotometer and diluted. The dilutions, which ranged from 10⁶ to 10¹ vector copy numbers, were used as standards in the quantitative RT-PCR (qRT-PCR) assays. The cloned fragments of 16S rDNA in the pGEM-BB and pGEM-LS vectors were amplified and sequenced with an automated sequence analyser (Genetic Analyser 3500, Applied Biosystems, CA, USA) using a 50-cm capillary array and a POP-7 polymer (Applied Biosystems) and the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, version 3.1) according to the manufacturer’s instructions. All electropherograms were manually edited for base ambiguity. The obtained FASTA sequences were aligned using CLUSTAL-W software (http://www.ebi.ac.uk/clustalw/) and used for the design of species-specific primers and TaqMan probes (Roche Diagnostics, Mannheim, Germany) (Supplementary Table 1).

Reddel S., Del Chierico F., Quagliariello A., Giancristoforo S., Vernocchi P., Russo A., Fiocchi A., Rossi P., Putignani L, & El Hachem M. (2019). Gut microbiota profile in children affected by atopic dermatitis and evaluation of intestinal persistence of a probiotic mixture. Scientific Reports, 9, 4996.

+ Open protocol

+ Expand

Cloning and Characterization of PrCYP707A1 Promoter

Check if the same lab product or an alternative is used in the 5 most similar protocols

The sequence of the PrCYP707A1 promoter was obtained using the GenomeWalker Universal Kit (Clontech, Palo Alto, CA, USA) according to manufacturer’s instructions. The promoter was amplified by a single PCR reaction using specific adaptor primers AP1 and a gene-specific primer GWPrCYP707A1 (Table 1). A single PCR product was cloned using a pGEM-T Easy Vector System kit (Promega, Madison, WI, USA) according to manufacturer’s instructions, and then sequenced (Eurofins MWG Operon, Ebersberg, Germany). A PrCYP707A1 promoter sequence of 2581bp was obtained and assembled with the PrCYP707A1 gene sequence (GenBank accession number: KM226163). Promoter motif searches were carried out using the PLACE software (Higo et al., 1999 (link)).

Lechat M.M., Brun G., Montiel G., Véronési C., Simier P., Thoiron S., Pouvreau J.B, & Delavault P. (2015). Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel. Journal of Experimental Botany, 66(11), 3129-3140.

+ Open protocol

+ Expand

Characterization of E. coli Strains

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following K12 E. coli strains were used: DH5α (Stratagene, USA), BL21 (DE3) (Novagen, USA) and C43 (DE3) (Lucigen, USA). The plasmid vector pET28a was obtained from Novagen (USA) and the pGEM-T Easy Vector System kit from Promega (USA). Bacterial isolates used in this study consisted of strains previously defined as ETEC by the presence of LT and/or ST encoding-gene, as well as the production of the respective toxins [18 (link)]. Also, ETEC H10407 (O78:H11) and 3321–4 (O153:H45) were employed as ST/LT-producing and ST-producing prototypes, respectively [19 (link), 20 (link)].

Ozaki C.Y., Silveira C.R., Andrade F.B., Nepomuceno R., Silva A., Munhoz D.D., Yamamoto B.B., Luz D., Abreu P.A., Horton D.S., Elias W.P., Ramos O.H, & Piazza R.M. (2015). Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli. PLoS ONE, 10(7), e0131484.

+ Open protocol

+ Expand

Cloning and Truncation of MIR31HG

Check if the same lab product or an alternative is used in the 5 most similar protocols

pGEM-MIR31HG plasmid was established by PCR amplification of MIR31HG sequence (NR_027054.2) from cDNA using primers listed in Supplementary Table 1 and cloned using TA cloning strategy with pGEM-T easy vector system kit (Promega), according to the manufacturer’s protocols. MIR31HG truncations were established by PCR amplification from pGEM-MIR31HG plasmid with primers indicated in Supplementary Table 1 and cloned with In-fusion HD cloning kit (Clontech).

Montes M., Lubas M., Arendrup F.S., Mentz B., Rohatgi N., Tumas S., Harder L.M., Skanderup A.J., Andersen J.S, & Lund A.H. (2021). The long non-coding RNA MIR31HG regulates the senescence associated secretory phenotype. Nature Communications, 12, 2459.

+ Open protocol

+ Expand

Plasmid Cloning and Quantitative PCR for Bifidobacterium Species

Check if the same lab product or an alternative is used in the 5 most similar protocols

The pGEM^®-T Easy Vector System kit (Promega, Milan, Italy) was used to clone the purified PCR fragment into the PGEM vector with the use of Escherichia coli competent cells. The extraction of recombinant plasmids pGEM-BB (pGEM+B. breve), pGEM-BL (pGEM+B. longum subsp. longum) and pGEM-BI (pGEM+B. longum subsp. infantis) was then performed (Plasmid Miniprep Kit, Promega, Italy). Obtained DNA was quantified using the NanoDrop ND-1000 spectrophotometer and serial dilutions of plasmids were prepared in the range from 10⁶ to 10¹ vector copy numbers and used to create a standard curve for the quantitative RT-PCR (qRT-PCR) assays. ITS rDNA cloned fragments were amplified and sequenced (Genetic Analyser 3500, Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. FASTA sequences were aligned with CLUSTAL-W software (http://www.ebi.ac.uk/clustalw/) (accessed on 1 February 2021) and species-specific primers and TaqMan probes (Roche Diagnostics) design was carried out (Supplementary Table S1).

Mennini M., Reddel S., Del Chierico F., Gardini S., Quagliariello A., Vernocchi P., Valluzzi R.L., Fierro V., Riccardi C., Napolitano T., Fiocchi A.G, & Putignani L. (2021). Gut Microbiota Profile in Children with IgE-Mediated Cow’s Milk Allergy and Cow’s Milk Sensitization and Probiotic Intestinal Persistence Evaluation. International Journal of Molecular Sciences, 22(4), 1649.

+ Open protocol

+ Expand

Comprehensive E. coli K12 Strain Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following E. coli K12 strains were used: DH10b (Stratagene, USA) and BL21 (DE3) pLyS (Novagen, USA). The plasmid vector pET28a (Novagen, USA) and the pGEM‐T Easy Vector System kit (Promega, USA) were used in order to construct the pGEM_uspF and pET28a_uspF plasmids, respectively. Bacterial isolates used in this study consisted of four atypical EPEC strains presenting different adhesion patterns, i.e., Ec292/84, 9100/83, BA320 and BA4013.29 Also, a collection of different bacterial pathogroups were analyzed for the presence of the uspF gene, i.e., typical EPEC (tEPEC), atypical EPEC (aEPEC), ETEC and STEC,17, 48, 49, 50, 51 as well as other Enterobacteriaceae isolates, including Morganella morganii, Klebsiella pneumoniae, Shigella boydii, Proteus mirabilis, Salmonella spp., and Citrobacter freundii. Further, groups of E. coli isolates that do not carry virulence factors found in diarrheagenic E. coli and belonging to our bacterial collection were also analyzed.

de Souza C.S., Torres A.G., Caravelli A., Silva A., Polatto J.M, & Piazza R.M. (2016). Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae. Protein Science : A Publication of the Protein Society, 25(12), 2142-2151.

+ Open protocol

+ Expand

Intestinal Eukaryotes Identification Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cloning and sequencing reactions were performed as previously described16 (link). The PCR products were cloned separately using the pGEM® -T Easy Vector System Kit (Promega, Madison, USA). Aliquots (150 µl) of cell suspensions were plated onto LB (Luria-Bertani Broth) agar plates that were supplemented with ampicillin (100 mg/mL), X-GAL (80 mg/mL) and IPTG (120 mg/mL), and the plates were incubated overnight at 37°C. Positive clones were suspended in 25 µL of distilled water and stored at −20°C. The presence of the insert was confirmed by PCR amplification using the M13 forward (5′-GTAAAACGACGGCCAG-3′) and M13 reverse (5′-AGGAAACAGCTATGAC-3′) primers (Eurogentec, Seraing, Belgium). The purified PCR products were sequenced in both directions using the BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing products were run on an ABI PRISM 3130 automated sequencer (Applied Biosystems). Finally, intestinal eukaryotes were identified by comparing the resulting sequences with those that were deposited in GenBank using the basic local alignment search tool (BLAST), which is available at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/). Phylogenetic analyses were performed using MEGA5.04 and a distance matrix neighbor-joining (NJ) approach57 (link).

Hamad I., Keita M.B., Peeters M., Delaporte E., Raoult D, & Bittar F. (2014). Pathogenic Eukaryotes in Gut Microbiota of Western Lowland Gorillas as Revealed by Molecular Survey. Scientific Reports, 4, 6417.

+ Open protocol

+ Expand

Herbicide Resistance Mutation Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

Multiple primer pairs were designed in order to cover all possible mutation points along the whole psbA and ahas gene sequences by using Primer3Plus [63] (link) (Figures S1 andS2).
In the case of intron containing epsps gene, the exon-intron bounds were predicted based on complete gene sequences of closely related species by using ClustalW aligning the sequences of Conyza canadensis (AY545667.1) and Amaranthus palmeri (FJ861242.1 and FJ861243.1) [64] (link) (Figure S3).
In order to validate polymorphisms that characterize herbicide resistance, the imazethapyr, linuron resistant, and sensitive bulked samples were amplified. The PCR amplifications was performed by using Dream Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) The PCR products were separated on 1.5% agarose gel (Promega, Madison, WI, USA) and were purified using NucleoSpin Gel and PCR Clean-up system (Macharey-Nagel GmbH & Co, Düren, Germany). The pGEM-T Easy Vector System kit (A1360 Promega, Madison, WI, USA) and JM109 Competent Cells were used to clone PCR products [65] . Sanger sequencing of the cloned fragments was performed with ABI 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Sequenced fragments of herbicide target genes were aligned to predicted gene sequences certifying amino acid substitutions in the mutation points.

Kutasy B., Farkas Z., Kolics B., Decsi K., Hegedűs G., Kovács J., Taller J., Tóth Z., Kálmán N., Kazinczi G., & Virág E. (2021). Detection of Target-Site Herbicide Resistance in the Common Ragweed: Nucleotide Polymorphism Genotyping by Targeted Amplicon Sequencing. Diversity, 13(3), 118-118.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!