A non-polar deletion of sslE was constructed in E. coli W and H10407 strains by allelic exchange with a FLP recombination target (FRT)-flanked Kanr cassette, using a modified protocol58 (link). The fragment was amplified from E. coli strain JW5925-137 using Platinum Taq DNA polymerase (Promega) and primers PC3 and PC4 and then exchange into E. coli W and H10407 strains were facilitated by the λ Red recombinase system carried on plasmid pKD46. E. coli strains carrying the pKD46 plasmid were grown in SOB media with 100 μg/ml ampicillin and 1 mM L-arabinose at 30 °C to an OD of 0.6. Cells were made electrocompetent, electroporated with 10–100 ng of PCR fragment and then selected on LB agar at 37 °C containing 25 μg/ml kanamycin, followed by transfer and growth on medium containing no antibiotics. Loss of the pKD46 plasmid was tested by ampicillin sensitivity. Knockouts were confirmed by PCR and sequencing.
Platinum taq dna polymerase
Manufactured by Promega
Sourced in United States
Platinum Taq DNA polymerase is a thermostable DNA polymerase enzyme used for DNA amplification in polymerase chain reaction (PCR) applications. It is designed to provide reliable and consistent performance in a wide range of PCR protocols.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Prl tk, by Promega (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dual glo system, by Promega (1 mentions)
Accutaq la dna polymerase, by Merck Group (1 mentions)
Mj mini, by Bio-Rad (1 mentions)
100 bp marker, by Thermo Fisher Scientific (1 mentions)
Bl21 star de3, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
6 protocols using platinum taq dna polymerase
1
Construction of sslE Deletion Mutants
2
Quantification of NHE-2 mRNA Expression
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An aliquot of total RNA [1μg] was mixed with the primers PR1 and PR2 [100 pmoles of each], buffer and 1 unit of Platinum Taq DNA polymerase supplied with the kit [Promega]. Reactions were performed as follows: Reverse transcription: 42°C x 45 min → 30 [Denaturation: 94°C x 30 sec, Annealing: 45°C x 30 sec, Extension: 74°C x 60 sec]. A standard curve between the cycle threshold [CT] and concentrations of purified CC was prepared separately. The Corresponding CT values were obtained for both the non colitis control and the colitis RNA samples. The level of NHE-2 mRNA was calculated using the CT curve. A change in the mRNA was calculated with respect to the control CT values.
3
Investigating lncRNA Regulation of ACACA and SREBF1
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lncACACA and lncSREBF1 cDNAs were amplified with high‐fidelity Platinum Taq DNA polymerase (Invitrogen) and inserted into pTracer‐SV40 (Thermo Fisher Scientific) using the ApaI and SpeI restriction sites. The ACACA and SREBF1 promoters were amplified from human genomic DNA with Platinum Taq DNA polymerase (primer sequence available on request) and inserted upstream to the firefly luciferase CDS in pGL4 (Promega) using the HindIII and XhoI restriction sites. HEK293 cells were first transfected with the lncRNA cDNA (2.5 μg plasmid) using Lipofectamine 3000 (Invitrogen) in 6‐well plates, as recommended by the manufacturer. At 24 hours later, cells were transfected with the promoter‐firefly luciferase construct (0.1 μg) in 96‐well plates. The Renilla luciferase vector (pRL‐TK; Promega) was cotransfected and used as expression control. Normalized expression was calculated as a firefly/Renilla luciferase activity ratio. Luciferase assays were performed in 96‐well chemoluminescence plates using the Dual‐Glo system (Promega), according to the manufacturer's instructions. Each assay was performed in triplicate.
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lncACACA and lncSREBF1 cDNAs were amplified with high‐fidelity Platinum Taq DNA polymerase (Invitrogen) and inserted into pTracer‐SV40 (Thermo Fisher Scientific) using the ApaI and SpeI restriction sites. The ACACA and SREBF1 promoters were amplified from human genomic DNA with Platinum Taq DNA polymerase (primer sequence available on request) and inserted upstream to the firefly luciferase CDS in pGL4 (Promega) using the HindIII and XhoI restriction sites. HEK293 cells were first transfected with the lncRNA cDNA (2.5 μg plasmid) using Lipofectamine 3000 (Invitrogen) in 6‐well plates, as recommended by the manufacturer. At 24 hours later, cells were transfected with the promoter‐firefly luciferase construct (0.1 μg) in 96‐well plates. The Renilla luciferase vector (pRL‐TK; Promega) was cotransfected and used as expression control. Normalized expression was calculated as a firefly/Renilla luciferase activity ratio. Luciferase assays were performed in 96‐well chemoluminescence plates using the Dual‐Glo system (Promega), according to the manufacturer's instructions. Each assay was performed in triplicate.
4
DNA Amplification Using DNA Polymerases
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DNA was amplified using Invitrogen Platinum Taq DNA Polymerase (High Fidelity), Promega GoTaq Colorless Master Mix, or Sigma-Aldrich Accutaq LA DNA Polymerase. All primers used in this work are shown in Table 1 .
5
Primer Design and Amplification of ps Genes
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Four specific sets of primers were designed using Primer3Plus
(https://primer3plus.com/cgi-bin/dev/primer3plus.cgi) software aligning ps gene sequences (PS1-PS4) (Okumura et al. 2010) . FASTA sequence of ps gene sequences was retrieved from NCBI database (Supplementary Table 2) and four specific primer set was designed (Supplementary Table 3). PCR for specific ps genes was carried out in a thermal cycler (MJ mini, Bio-Rad) using both genomic and plasmid DNA in total 50 µl reaction mixture including 5 µl of 10× buffer, 1.0 µl 10 mM dNTPs, 2.0 µl MgSO 4 , 1.0 µM of each primer, 20-50 ng DNA template, 0.25 µl Platinum Taq DNA polymerase (Promega, USA) and 2 µl DMSO. Amplification was achieved by 35 cycles (96 °C for the 50s, 47 °C (ps1, ps2, and ps3) and 50 °C (ps4) for 45s, 72
°C for 2 min) with an initial denaturation step at 96 °C for 10 min and a final extension step at 72 °C for 10 min and the amplicons were analyzed by electrophoresis in 1.5% agarose gel along with 100 bp marker (Invitrogen, USA).
(https://primer3plus.com/cgi-bin/dev/primer3plus.cgi) software aligning ps gene sequences (PS1-PS4) (Okumura et al. 2010) . FASTA sequence of ps gene sequences was retrieved from NCBI database (Supplementary Table 2) and four specific primer set was designed (Supplementary Table 3). PCR for specific ps genes was carried out in a thermal cycler (MJ mini, Bio-Rad) using both genomic and plasmid DNA in total 50 µl reaction mixture including 5 µl of 10× buffer, 1.0 µl 10 mM dNTPs, 2.0 µl MgSO 4 , 1.0 µM of each primer, 20-50 ng DNA template, 0.25 µl Platinum Taq DNA polymerase (Promega, USA) and 2 µl DMSO. Amplification was achieved by 35 cycles (96 °C for the 50s, 47 °C (ps1, ps2, and ps3) and 50 °C (ps4) for 45s, 72
°C for 2 min) with an initial denaturation step at 96 °C for 10 min and a final extension step at 72 °C for 10 min and the amplicons were analyzed by electrophoresis in 1.5% agarose gel along with 100 bp marker (Invitrogen, USA).
6
Cloning and Expression of T. cruzi GALK Genes
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Sequences of the T. cruzi GALK genes (accession codes TcCLB. 507001.110 and TcCLB.510667.120) were retrieved from the GeneDB database (http://www.Genedb.org/). Specific primers were designed in order to amplify by PCR both full-length genes: for TcGALK-1 the forward primer 5´-CGCATATGAATCCCCTCAGCTACAC-3´(NdeI site in bold) and the reverse primer 5´-GCGGATCCCTACAGGTTGCTATCGGGC-3´(BamHI site in bold), for TcGALK-2 as forward primer 5′-CGCATATGCCGAG CTACTCAGACAA-3′ (NdeI site in bold) and as reverse primer 5′-CGGGATCCTCATAGCTTTAACACCC-3′ (BamHI site in bold). PCR was performed using the above-mentioned primers, Platinum Taq DNA polymerase (Promega), and genomic DNA from T. cruzi (EP strain). In both cases, the PCR product was ligated into the pGEM-T-easy vector (Promega), and sequenced using the T7 and SP6 universal primers in an automated sequencer. Subsequently, each full-length gene was transferred to the pET28a vector (Novagen) and used to transform E. coli strains BL21(DE3) pLys (Novagen) and BL21 Star (DE3) (Life Technologies). The expression systems yielded recombinant proteins contained a Nterminal 20-residues long extension having a (His) 6 -tag plus a cleavage site for thrombin protease.
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