The TransformAid Bacterial Transformation Kit (Fermentas, USA) was used to transform Escherichia coli (E.coli) strains BL21(DE3) and DH5α with iNOS and CaM plasmid DNAs either separately or together using the expression vector pCW through standard procedures [12 (link)]. The transformed bacteria were then grown overnight in cultures and stored at -80°C as glycerol stocks for further use.
Transformaid bacterial transformation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
The TransformAid Bacterial Transformation Kit is a laboratory equipment designed for the transformation of competent bacterial cells. It facilitates the introduction of plasmid DNA into the cells, enabling genetic modification and expression studies.
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Lab products found in correlation
Clonejet pcr cloning kit, by Thermo Fisher Scientific (4 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Pcr cloning kit, by Qiagen (1 mentions)
Ptz57r t, by Thermo Fisher Scientific (1 mentions)
Qiamp dna stool kit, by Qiagen (1 mentions)
Topo ta vector, by Thermo Fisher Scientific (1 mentions)
Genelute gel extraction kit, by Merck Group (1 mentions)
Abi 3500 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Sequencing buffer, by Thermo Fisher Scientific (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
Bsteii, by Thermo Fisher Scientific (1 mentions)
Ecori, by New England Biolabs (1 mentions)
15 protocols using transformaid bacterial transformation kit
1
Bacterial Transformation for Protein Expression
2
Isolation and Sequencing of Satellite DNA from Pseudis and Lysapsus Species
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Genomic DNA samples were obtained from liver tissue from male and female individuals of Pseudis and Lysapsus species (Supplementary Table 1 ) following the TNES method employed by Medeiros et al. (2013) (link). Integrity of genomic DNA was evaluated by electrophoresis in a 0.8% agarose gel and total genomic DNA was quantified in a Nanodrop (Thermo Fisher, United States) spectrophotometer. PcP190 satDNA was isolated by PCR using the primers P190F (5′-AGACTGGCTGGGAATCCCAG-3′) and P190R (5′-AGCTGCTGCGATCTGACAAGG-3′), described previously by Vittorazzi et al. (2011) (link). The PCR program used was: (1) 94°C for 8 min; (2) 39 cycles of 94°C for 30 s, 58°C for 1 min and 72°C for 2.5 min; (3) 72°C for 8 min. PCR amplicons were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) and inserted in a plasmid pGEM-T Easy Vector (Promega). Plasmids were cloned into E. coli JM-109 bacteria using the TransformAid Bacterial Transformation Kit (Fermentas), according to manufacturer instructions. Recombinant colonies were selected, plasmid DNA was extracted following Sambrook et al. (1989) , and the inserts were amplified by PCR using the T7 and SP6 universal primers. After purification with the Wizard SV Gel and PCR Clean-Up System (Promega), the cloned fragments were sequenced on both strands using BigDye Terminator (Applied Biosystems) according to manufacturer instructions.
3
Cloning and Confirmation of PCR Amplicons
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Cloning of the PCR amplicon(s) for the genomic region as described above has been performed using pDrive cloning vector (Qiagen PCR Cloning Kit, Catalog number 231124). DH5α E. coli cell was used for transformation of the plasmid using Transform Aid Bacterial Transformation Kit (Fermentas, Catalog number K2711). Subsequently, clones were confirmed by clones' confirmation PCR.
4
Phage Binding Motif Modification
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Modification of the phage binding motif was achieved by standard cloning procedures. The corresponding oligonucleotides were annealed (cf. Table 1 ) and separately ligated into Acc65I / EagI cleaved M13KE-phage DNA. Ligated DNA was transformed into E. coli ER2738 using the TransformAid bacterial transformation kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. Positive clones were identified using LB/S-Gal/IPTG-plates. Following amplification, phage DNA was extracted using the E.Z.N.A. plasmid miniprep kit I (peqLab, Erlangen, Germany) and inserts were verified by DNA sequencing.
5
Amplification and Cloning of P450 Fragments
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Degenerate primers: forward P450F (5′-GARACIYTIMGNAARTAYCC-3′) and reverse P450R (5′-ATRCADATICKIGGNCCYTC-3′) based on motif of family 6 P450 were used to generate partial sequence of family 6 P450 fragments (
Huang et al. 2008 (link)
). PCR was carried out with 94°C initial denaturation for 3 min, 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The reaction mixture (50 μl) contained 2 μl of first-strand cDNA, 7 μM of each primer, 0.2 mM dNTP (deoxyribonucleotide) mix, 2.5 U
TaqDNA polymerase recombinant (Fermentas, Glen Burnie, MD), 1×
Taqbuffer with KCl, and 1.75 mM MgCl
2. Amplicons of ∼250 bp were purified and ligated into pTZ57R/T (Fermentas, Glen Burnie, MD) and cloned using TransformAid Bacterial Transformation Kit (Fermentas, Glen Burnie, MD). Plasmids purified from 10 white colonies were sent for sequencing. Sequences obtained were analyzed to verify the presence of P450 consensus region. Gene-specific primers for RACE based on these sequences were designed using Primer3Plus (
Untergasser et al. 2007
).
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Degenerate primers: forward P450F (5′-GARACIYTIMGNAARTAYCC-3′) and reverse P450R (5′-ATRCADATICKIGGNCCYTC-3′) based on motif of family 6 P450 were used to generate partial sequence of family 6 P450 fragments (
Huang et al. 2008 (link)
). PCR was carried out with 94°C initial denaturation for 3 min, 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The reaction mixture (50 μl) contained 2 μl of first-strand cDNA, 7 μM of each primer, 0.2 mM dNTP (deoxyribonucleotide) mix, 2.5 U
TaqDNA polymerase recombinant (Fermentas, Glen Burnie, MD), 1×
Taqbuffer with KCl, and 1.75 mM MgCl
2. Amplicons of ∼250 bp were purified and ligated into pTZ57R/T (Fermentas, Glen Burnie, MD) and cloned using TransformAid Bacterial Transformation Kit (Fermentas, Glen Burnie, MD). Plasmids purified from 10 white colonies were sent for sequencing. Sequences obtained were analyzed to verify the presence of P450 consensus region. Gene-specific primers for RACE based on these sequences were designed using Primer3Plus (
Untergasser et al. 2007
).
6
Microbiome DNA Extraction and Sequencing
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From a sample of 200 μL of the homogenate of the complete intestinal content containing the proximal and distal intestine (1:1), bacterial DNA was extracted using the QIAMP DNA Stool kit (QIAGEN), according to the manufacturer's guidelines. Bacterial DNA was verified by the amplification of a fragment of rRNA 16S gene using the 27 F and 907R universal primers, as was previously described and visualized with 1 % agarose gels. PCR products were purified from agarose gels with the Wizard SV Gel kit and PCR Clean-up System (Promega) and cloned using the TOPO TA vector according to the procedures indicated by Invitrogen. Cultures of Escherichia coli JM 107 strain were made competent using the Transform Aid Bacterial Transformation Kit (Fermentas), following the manufacturer's guidelines. Each clone was picked and cultured in LB broth with ampicillin for 16 h. To isolate plasmidic DNA with the insert, 100 μL of liquid culture was centrifuged at 6000g for 30 min, the medium was discarded, and the pellet was resuspended in 100 μL of sterile water, incubated at 95 °C for 30 min to produce cellular lysis, and then centrifuged at 6000g for 30 min. Finally, 5 μL of the lysate was amplified to detect the occurrence of the insert, using the M13F and M13 R primers. PCR products were verified in 1 % agarose gels, purified and sequenced by Macrogen (Seoul, Korea).
7
Cloning and Characterization of Glutaminase Gene
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Geobacillus thermodenitrificans DSM-465 glutaminase gene comprises 930 bp open reading frame (Accession no. NC 009328). Forward and reverse primers with 5′-catatggggtttcaaggtaacggtgtcac-3′ and 5′-atggatccttaagccggacgcaaatg-3′ were used for PCR amplification of gene. The reaction mixture containing 2.5 mM MgCl2, 2.0 mM dNTPs, 40 picomole primers, 15–20 ng of template DNA, and 2.5 U Taq DNA polymerase was subjected to thermocycler conditions of 94 °C, 63 °C and 72 °C for 35 cycles. The PCR-amplified glutaminase gene was analysed via agarose gel electrophoresis and purified using the GenElute™ Gel Extraction Kit (Sigma-Aldrich) according to the procedure described by the supplier. The purified PCR product was ligated into pTZ57R/T cloning vector; ligation mixture was incubated overnight at 22 °C and used for the transformation of bacterial cells. The TransformAid bacterial transformation kit (Thermo Fisher Scientific, catalogue no. k2710) was used for the transformation of bacterial competent cells with the recombinant plasmid. The successfully transformed colonies were screened for the presence of glutaminase gene by isolation and restriction of plasmids with NdeI and BamHI followed by agarose gel electrophoresis. The gene was subcloned in the pET21a (+) plasmid, and recombinant E. coli cells were obtained by plasmid isolation and restriction analysis, as abovementioned.
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Geobacillus thermodenitrificans DSM-465 glutaminase gene comprises 930 bp open reading frame (Accession no. NC 009328). Forward and reverse primers with 5′-catatggggtttcaaggtaacggtgtcac-3′ and 5′-atggatccttaagccggacgcaaatg-3′ were used for PCR amplification of gene. The reaction mixture containing 2.5 mM MgCl2, 2.0 mM dNTPs, 40 picomole primers, 15–20 ng of template DNA, and 2.5 U Taq DNA polymerase was subjected to thermocycler conditions of 94 °C, 63 °C and 72 °C for 35 cycles. The PCR-amplified glutaminase gene was analysed via agarose gel electrophoresis and purified using the GenElute™ Gel Extraction Kit (Sigma-Aldrich) according to the procedure described by the supplier. The purified PCR product was ligated into pTZ57R/T cloning vector; ligation mixture was incubated overnight at 22 °C and used for the transformation of bacterial cells. The TransformAid bacterial transformation kit (Thermo Fisher Scientific, catalogue no. k2710) was used for the transformation of bacterial competent cells with the recombinant plasmid. The successfully transformed colonies were screened for the presence of glutaminase gene by isolation and restriction of plasmids with NdeI and BamHI followed by agarose gel electrophoresis. The gene was subcloned in the pET21a (+) plasmid, and recombinant E. coli cells were obtained by plasmid isolation and restriction analysis, as abovementioned.
8
Cloning and Sequencing of MHC Amplicons
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PCR products were purified using a GeneJet Gel Extraction Kit (Thermo Scientific™), and the purified amplicons were cloned into the pJET vector using the CloneJET PCR cloning kit, both according to the manufacturer’s guidelines (Thermo Scientific™). Next, the cloned amplicons were transformed in Escherichia coli XL1-blue cells by using the TransformAid Bacterial Transformation Kit (Thermo Scientific™). Per animal, 24 to 48 bacterial clones were picked, and plasmid DNA was isolated using a standard mini-preparation procedure. The purified plasmid DNA was sequenced on the ABI 3500 genetic analyser (Applied Biosystems, Foster City, USA). The sequencing reaction was performed by using 2 μM pJET primer, 1 μL BigDye terminator, and 2 μL of 5 × sequencing buffer in a total volume of 10 μL (Thermo Scientific™). The resulting sequences were analysed using the Sequence Navigator programme (Applied Biosystems, Foster City, USA). MHC sequences were revised manually by applying the Lasergene 12 SeqMan Pro Sequence Alignment Editor.
9
Resolving Sequence Ambiguities through Cloning
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Molecular cloning was used to resolve double peaks in Sanger sequences and/or to confirm sequences obtained from amplicon sequencing in certain cases. Corresponding PCR products were cloned with the CloneJet™ PCR cloning kit (Fermentas) and the TransformAid Bacterial Transformation Kit (Thermo Scientific) according to manufacturer’s instructions. Individual colonies were transferred directly into the PCR mix. DNA was extracted during initial denaturation and served as a template for amplification with primers given in Table 7 . The PCR product obtained was sequenced by standard Sanger sequencing. Maximum of 6 colonies were used for PCR amplifications and sequencing.
10
Cloning and Sequencing of PR and COI Genes from R. nigrum
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The amplified R. nigrum cv. Didikai cDNA fragments were excised from agarose gel and purified using the GeneJET Gel Extraction Kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer’s protocol. Fragments were ligated into the pJET 1.2 blunt vector using the CloneJET™ PCR Cloning Kit (Thermo Scientific, Vilnius, Lithuania). Bacteria Escherichia coli JM107 were transformed with the TransformAid Bacterial Transformation Kit (Thermo Scientific, Vilnius, Lithuania). A total of 20 plasmids with cDNA inserts of PR and COI homologs were prepared for sequencing using the Big Dye Terminator v 3.1 Cycle Sequencing Kit and performed on a 3130 Genetic Analyzer Gene Analyzer (Applied Biosystem, Waltham, MA, USA). The sequenced 7 PR and 4 COI nucleotide sequences from R. nigrum were submitted to the NCBI database, and the accession numbers OK625407–OK625413 and OK625547–OK625550, respectively, were assigned (Table S5 ).
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