Brugmansia x candida was collected at the Nawarat Botanical Garden in Pathumthani, Thailand. This study was carried out on private land with the permission from the owner of the land. No specific permissions were required for this location/activity. This study did not involve endangered or protected species. The plant was identified by botanist at the Queen Sirikit Botanic Garden Herbarium. The voucher specimen was prepared and deposited in the Queen Sirikit Botanic Garden Herbarium. Total RNAs were isolated from leaves, stems, and flowers of B. x candida by using InviTrap® spin plant RNA mini-kit (STRATEC, Germany). Plant defensin cDNA was cloned by a two-step reverse transcriptase polymerase chain reaction (RT-PCR) using the degenerated primer SolaDefF1 [F: 5'-ATGGCACA(A/C)TC(T/C)AT(GTC)CGTTT(G/C)TTTGC-3' ] and oligo dT. The PCR conditions were: 98°C for 30 sec, followed by 35 cycles of amplification (98°C for 10 sec, 52°C for 30 sec, 72°C for 45 sec) and final extended at 72°C for 10 min. The PCR-amplified products were purified by E.Z.N.A. Gel Extraction Kit (Omega Biotek, USA) and cloned into pTG19-T vector (Vivantis, Malaysia). The positive clones were identified through restriction digest with BamHI and the sequences were determined using M13F forward primer. The nucleotide sequence of BcDef was submitted to GenBank under accession number MG923958.
Ptg19 t vector
Manufactured by Vivantis Technologies
Sourced in Malaysia
The PTG19-T vector is a laboratory equipment product used for molecular biology applications. It serves as a cloning vector, allowing the insertion and expression of DNA fragments. The core function of the PTG19-T vector is to facilitate the manipulation and study of genetic material in controlled experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Invitrap spin plant rna mini kit, by Stratec (1 mentions)
E z n a gel extraction kit, by Omega Bio-Tek (1 mentions)
E coli dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
Pgem 7zf vector, by Promega (1 mentions)
T4 dna ligase, by Vivantis Technologies (1 mentions)
Bamhi enzyme, by Thermo Fisher Scientific (1 mentions)
Dh5α competent cells, by Takara Bio (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Viral rna extraction kit, by Bioneer (1 mentions)
8 protocols using ptg19 t vector
1
Cloning of Plant Defensin from Brugmansia x candida
2
Aptamer Screening and Selection
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Aptamers obtained from the last round of SELEX were amplified by PCR and were cloned into a pTG19-T vector (Vivantis, Selangor, Malaysia). The ligation reaction was transformed into E. coli DH5α competent cells (Thermo Fisher Scientific, Waltham, MA USA) and plated on LB agar containing 50 μg.ml−1 ampicillin. Colonies were analyzed with colony PCR. Each positive clone was amplified with FITC-labeled primers and the fluorescence intensity was estimated by flow cytometer. Then aptamer candidates with the highest binding affinities were selected for further analysis.
3
Evaluating Primer Specificity for miR-326 and RNU6
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The specificity of primers was evaluated by running miR-326 and RNU6 real-time PCR products on 12% poly acrylamide gel to see the solo amplified band. The resultant electrophoresis bands were then T/A cloned into pTG19-T vector (Vivantis, Malaysia) and sent for sequencing.
4
Isolation and Characterization of Iranian Lizard Leishmania
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Iranian Lizard Leishmania (I.L.L) isolated by Kazemi et al. (11 ) was present in the biotechnology lab. pGEM-7zf vector (Promega Company, USA) and pTG19-T vector (Vivantis, Malaysia) were purchased.
5
Cloning and Sequencing of RT-PCR Amplicons
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Amplified fragments resulting from the reverse transcription polymerase chain reaction (RT-PCR) were ligated into the pTG19-T vector (Vivantis, Malaysia) using 1 Unit of T4 DNA ligase (Vivantis, Malaysia) according to the manufacturer’s protocol and incubated at 4°C overnight. Ligation mixes were transformed into prepared chemically competent Escherichia coli strain DH5α cells by heat shock method (Chung et al., 1989 (link)). The transformed cells were selected on LB plates containing ampicillin (100 mg/ml), IPTG (100 μl of 0.1 M per plate), and X-Gal (20 μl of 50 mg/ml). Plasmids were extracted by Accuprep nanopluse plasmid mini extraction kit (Bioneer, South Korea) according to manufacturer’s instruction. The recombinant plasmids were verified by PCR colony and each colony carrying the cloned cDNA was subjected to single colony isolation. Also, the purified plasmids were digested by BamHI enzyme (Thermo Fisher Scientific, USA) to release the DNA inserted and loaded on 1% agarose gel. Next, the independent clones of each viral isolate were subjected to nucleotide sequencing with M13F/R primers. The sequencing was done by Bioneer Inc. (Seoul, South Korea).
6
BEFV G Gene Amplification Protocol
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BEFV G gene was amplified as described by Hsieh et al with some modification [32 (link)]. A nested PCR was designed according to primers introduced by Hsieh et al. In the first run, G1F and G4R primers were used to amplify an 1872 length fragment with the following protocol: 94 °C for 2 min, 25 cycles of 94 °C for 50 s, 50 °C for 50 s, 72 °C for 75 s, and a final extension of 72 °C for 5 min. In the second run, the PCR product was used as a template and G1-G4 fragments were amplified. PCR products were directly sequenced or subcloned into the pTG19-T vector (Vivantis; Malaysia) using standard techniques and then sequenced again. Sequencing was performed by Bioneer Company (South Korea) with the same PCR primers in two directions. Sequences were trimmed with BioEdit software version 7.0.4.1 (mbio, Inc., North Carolina, USA). Obtained sequences were submitted to the GenBank and are available under the accession numbers MZ51169 and MZ51168.
7
Cloning and Sequencing of linc-ROR
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All linc-ROR PCR products were purified from agarose
gel with ExpinTM combo kit (GeneAll, South Korea). Then,
PTG19-T vector (Vivantis, Malaysia) was applied to clone
the purified products using T4 DNA ligase (Fermentas, USA)
and transformed to DH5α competent cells (TaKaRa, Japan).
Recombinant colonies with resistance to ampicillin
(Sigma-Aldrich, USA) were selected as positive
ones. Universal M13 primers were employed to select
different variants of the linc-ROR gene via colony
check PCR. Sanger sequencing (Microgen, South
Korea) was utilized to validate identity and validity of
the PCR products.
gel with ExpinTM combo kit (GeneAll, South Korea). Then,
PTG19-T vector (Vivantis, Malaysia) was applied to clone
the purified products using T4 DNA ligase (Fermentas, USA)
and transformed to DH5α competent cells (TaKaRa, Japan).
Recombinant colonies with resistance to ampicillin
(Sigma-Aldrich, USA) were selected as positive
ones. Universal M13 primers were employed to select
different variants of the linc-ROR gene via colony
check PCR. Sanger sequencing (Microgen, South
Korea) was utilized to validate identity and validity of
the PCR products.
8
BEFV G Gene Amplification and Sequencing
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RNA was extracted from buffy coats using a viral RNA extraction kit (Bioneer; South Korea) according to the manufacturer's protocol. Then, extracted RNA samples were reverse-transcribed using a kit (Yekta Tajhiz; Iran) to obtain cDNA. Screening of the positive BEFV samples was performed based on the ampli cation of the partial G gene via a nested PCR [30, 31] . Primer sequences are indicated in Table 2.
Sequencing the full-length G gene BEFV G gene was ampli ed as described by Hsieh et al with some modi cation [32] . A nested PCR was designed according to primers introduced by Hsieh et al. In the rst run, G1F and G4R primers were used to amplify an 1872 length fragment with the following protocol: 94 C for 2 min, 25 cycles of 94 C for 50 s, 50 C for 50 s, 72 C for 75 s, and a nal extension of 72 C for 5 min. In the second run, the PCR product was used as a template and G1-G4 fragments were ampli ed. PCR products were directly sequenced or subcloned into the pTG19-T vector (Vivantis; Malaysia) using standard techniques and then sequenced again. Sequencing was performed by Bioneer Company (South Korea) with the same PCR primers in two directions. Sequences were trimmed with BioEdit software version 7.0.4.1 (mbio, Inc, North Carolina, USA). Obtained sequences were submitted to the GenBank and are available under the accession numbers MZ51169 and MZ51168.
Sequencing the full-length G gene BEFV G gene was ampli ed as described by Hsieh et al with some modi cation [32] . A nested PCR was designed according to primers introduced by Hsieh et al. In the rst run, G1F and G4R primers were used to amplify an 1872 length fragment with the following protocol: 94 C for 2 min, 25 cycles of 94 C for 50 s, 50 C for 50 s, 72 C for 75 s, and a nal extension of 72 C for 5 min. In the second run, the PCR product was used as a template and G1-G4 fragments were ampli ed. PCR products were directly sequenced or subcloned into the pTG19-T vector (Vivantis; Malaysia) using standard techniques and then sequenced again. Sequencing was performed by Bioneer Company (South Korea) with the same PCR primers in two directions. Sequences were trimmed with BioEdit software version 7.0.4.1 (mbio, Inc, North Carolina, USA). Obtained sequences were submitted to the GenBank and are available under the accession numbers MZ51169 and MZ51168.
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