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One shot max efficiency dh5α t1r competent cells

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One Shot MAX Efficiency DH5α-T1R competent cells are a strain of Escherichia coli bacteria that have been genetically modified to be highly competent for DNA transformation. These cells are optimized for efficient uptake and integration of foreign DNA, making them a useful tool in molecular biology and genetic engineering applications.

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Lab products found in correlation

Geneart site directed mutagenesis plus system, by Thermo Fisher Scientific (1 mentions) Accuprime pfx dna polymerase, by Thermo Fisher Scientific (1 mentions) Accuprime taq dna polymerase, by Thermo Fisher Scientific (1 mentions) Minelute gel purification kit, by Qiagen (1 mentions) Pcr4 topo, by Thermo Fisher Scientific (1 mentions) Nanodrop 200, by Thermo Fisher Scientific (1 mentions) Soc media, by Thermo Fisher Scientific (1 mentions) Ampicillin, by Merck Group (1 mentions) Purelink hipure plasmid midiprep kit, by Thermo Fisher Scientific (1 mentions) Geneart site directed mutagenesis plus kit, by Thermo Fisher Scientific (1 mentions)

5 protocols using one shot max efficiency dh5α t1r competent cells

1

Purification and Mutagenesis of σ1 Protein

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Construct σ1long, comprising the three most C-terminal β-spirals of T1L σ1 and the entire head domain (amino acids 261 to 470), was purified as described previously (22 (link)) and used in STD-NMR experiments. Mutations were generated using the GeneArt site-directed mutagenesis PLUS system (Invitrogen) along with AccuPrime Pfx DNA polymerase (Invitrogen) and One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen).
Stencel-Baerenwald J., Reiss K., Blaum B.S., Colvin D., Li X.N., Abel T., Boyd K., Stehle T, & Dermody T.S. (2015). Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus. mBio, 6(2), e02356-14.
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2

PCR-based Transgene Verification

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PCRs were performed using AccuPrime Taq DNA Polymerase, High Fidelity (Invitrogen, LS12346086) and primers (see S1 Table) that would only generate an amplicon if the Gfp-GPI transgene insert was present. PCR products were extracted from agarose gels using a MinElute Gel Purification Kit (Qiagen, 28604), ligated into the pCR2.1–TOPO TA vector using a cloning kit (Invitrogen, 450641) and transformed into One Shot Max Efficiency DH5α-T1R Competent Cells (Invitrogen, 12297016). Plasmid DNA samples were subjected to diagnostic restriction digests followed by Sanger sequencing.
Castle A.R., Wohlgemuth S., Arce L, & Westaway D. (2022). Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles. PLoS ONE, 17(6), e0269342.
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3

Cloning and Sequencing of PCR Products

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The expected PCR bands were examined on a 1% agarose gel and then purified. The purified PCR product was cloned into pCR4®TOPO (Invitrogen Carlsbad, CA) and transformed into One Shot® MAX Efficiency® DH5α™-T1R Competent Cells (Invitrogen, Carlsbad, CA). Positive clones were confirmed by sequencing using an ABI Prism 3100 Applied Biosystems Sequencer (CA, USA). The sequences were analyzed using BioEdit Sequence Alignment Editor and CLC Main Workbench 6 (http://www.clcbio.com).
Zapata M., Kunii I.S., Paninka R.M., Simões D.M., Castillo V.A., Reche A J.r., Maciel R.M, & Dias da Silva M.R. (2014). Molecular cloning of ion channels in Felis catus that are related to periodic paralyses in man: a contribution to the understanding of the genetic susceptibility to feline neck ventroflexion and paralysis. Biology Open, 3(9), 785-793.
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4

Efficient Transformation of E. coli with Plasmid

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One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen, Massachusetts, USA) were incubated with 1 µl of plasmid on ice for 30 min. These cells were then heat shocked at 42 °C for 30 s, returned to ice for 2 min, and grown in 950 µl SOC media (Invitrogen, Massachusetts, USA) at 37 °C with shaking for 1 h. Resulting transformed Escherichia coli cells were plated on LB agar with ampicillin (100 µg/ml; Sigma Aldrich, Missouri, USA) and grown over night at 37 °C. Colonies were then grown 8 h in 10 ml LB medium with ampicillin (100 µg/ml) followed by growing 400 ml to extract plasmids. Transfection-grade plasmid DNA was obtained using the NucleoBond Xtra Midi or Maxi kits (Macherey–Nagel, Düren, Germany). Final DNA product was assessed for quantity and quality using NanoDrop 200- (Thermo Fisher Scientific, Massachusetts, USA).
Pollak N.M., Cooper-White J.J, & Macdonald J. (2021). Translational control of enzyme scavenger expression with toxin-induced micro RNA switches. Scientific Reports, 11, 2462.
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5

Site-Directed Mutagenesis of F13A Plasmid

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We used the pcDNA5/FRT expression plasmid containing the human wild-type F13A cDNA sequence (pcDNA5/FRT-WT-F13A) we had cloned earlier9 (link). The mutant F13A expression plasmid with a mutation in exon 2 at codon 36 (c.109C>T, Pro36Ser) was constructed by site-directed mutagenesis of the wild-type expression plasmid (pcDNA5/FRT-WT-F13A) using the GeneArt® Site-Directed Mutagenesis PLUS Kit (Invitrogen). The mutagenic primers were designed using the web-based GeneArt® Primer and Construct Design Tool (http://www.thermofisher.com/order/oligoDesigner). The following primers were synthesised by Microsynth: forward primer 5’-CTTCAGGGCGTGGTGTCCCGGGGCGTCAACC-3’, and reverse primer 5’-GGTTGACGCCCCGGGACACCACGCCCTGAAG-3’. The mutagenesis reaction was performed according to the manufacturer’s instruction using One Shot® MAX Efficiency® DH5α™-T1R competent cells (Invitrogen). Plasmids were purified using the PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen) and then sequenced by Microsynth using their standard CMV-forward and BGH-reverse primers. The plasmids with correct wild-type (pcDNA5/FRT-WT-F13A) and mutant (pcDNA5/FRT-MU-F13A) sequences were used for transfection.
Li B., Billur R., Maurer M.C., Kohler H.P., Raddatz Müller P., Alberio L, & Schroeder V. (2018). Proline 36 of the Factor XIII Activation Peptide Plays a Crucial Role in Substrate Recognition and Zymogen Activation. Thrombosis and haemostasis, 118(12), 2037-2045.
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