One shot max efficiency dh5α t1 competent cells

Manufactured by Thermo Fisher Scientific

The One Shot Max Efficiency DH5α-T1 Competent Cells are a high-efficiency competent cell line designed for transformation of plasmid DNA. These cells are suitable for general cloning applications and offer reliable performance in bacterial transformation experiments.

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Lab products found in correlation

Qiaquick pcr purification kit, by Qiagen (2 mentions) Qiaquick gel extraction kit, by Qiagen (2 mentions) Quick ligation kit, by New England Biolabs (2 mentions) Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions) Smarter race kit, by Takara Bio (2 mentions) Advantage 2 pcr kit, by Takara Bio (2 mentions) μmacs oligo dt microbeads, by Miltenyi Biotec (2 mentions) Nucleospin extract 2 gel extraction kit, by Macherey-Nagel (2 mentions) Rnalater, by Thermo Fisher Scientific (2 mentions) Superscript 2, by Thermo Fisher Scientific (2 mentions) Facsaria 3, by BD (2 mentions) Hotstar hifidelity pcr kit, by Qiagen (1 mentions) Geneart site directed mutagenesis plus system, by Thermo Fisher Scientific (1 mentions) Fastdigest dpni, by Thermo Fisher Scientific (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)

Close spelling variants of this product

MAX Efficiency DH5α Competent Cells One ShotTM competent cells MAX Efficiency DH5α DH5α competent cells DH5α cells

6 protocols using one shot max efficiency dh5α t1 competent cells

Cloning of MRS6 Gene into Yeast Vectors

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The MRS6 gene was cloned into the pGAD-C1 and pGBDU-C1 vectors in the following way. The MRS6 gene was amplified by PCR using the forward primer 5′-ATGCATCGATATGTTAAGTCCTGAACGTAGACC-3′ and reverse primer 5′-ATGCGTCGACTCATATCTCCATTTCACCTACAAATTC-3′. The PCR product was purified with QIAquick PCR Purification Kit, Qiagen, CA#28106. The PCR product and pGAD-C1 vector were digested with ClaI (5′-ATCGAT-3′, New England BioLabs Inc., MA, CA#R0197S) and SalI (5′-GTCGAC-3′, New England Biolabs Inc., MA, CA#R3138S) restriction enzymes. Digested insert and vector DNAs were run on a 1% agarose gel containing ethidium bromide. Bands were extracted from the gel using the QIAquick Gel Extraction Kit, Qiagen (CA#28704). A quick Ligation Kit (New England Biolabs Inc., MA, CA#M200l) was used for ligating the insert and vector. The ligation mixture was transformed into E. coli (One-Shot MAX Efficiency DH5α-T1 Competent Cells, ThermoFisher, CA# 12297016), followed by plating on LB + Carb plates. The plates were incubated at 37 °C for 24 h. Transformants were confirmed by digestion with ClaI and SalI. Plasmids were sequenced at the Roswell Park Sequencing facility (Roswell Park Cancer Institute, Buffalo, NY).

Jamalzadeh S., Pujari A.N, & Cullen P.J. (2020). A Rab escort protein regulates the MAPK pathway that controls filamentous growth in yeast. Scientific Reports, 10, 22184.

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Cloning MRS6 gene into yeast vectors

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The MRS6 gene was cloned into the pGAD-C1 and pGBDU-C1 vectors in the following way.
The MRS6 gene was amplified by PCR using the forward primer 5'-ATGCATCGATATGTTAAGTCCTGAACGTAGACC-3' and reverse primer of 5'-ATGCGTCGACTCATATCTCCATTTCACCTACAAATTC-3'. The PCR product was purified with QIAquick PCR Purification Kit, Qiagen, CA#28106. The PCR product and pGAD-C1 vector were digested with ClaI (5'-ATCGAT-3', New England BioLabs Inc., MA, CA#R0197S) and SalI (5'-GTCGAC-3', New England Biolabs Inc., MA, CA#R3138S) restriction enzymes.
Digested insert and vector DNAs were run on a 1% agarose gel containing ethidium bromide.
Bands were extracted from the gel using the QIAquick Gel Extraction Kit, Qiagen (CA#28704).
A quick Ligation Kit (New England Biolabs Inc., MA, CA#M200l) was used for ligating the insert and vector. The ligation mixture was transformed into E. coli (One-Shot MAX Efficiency DH5α-T1 Competent Cells, ThermoFisher, CA# 12297016), followed by plating on LB+Carb plates. The plates were incubated at 37°C for 24 h. Transformants were confirmed by digestion with ClaI, and SalI Plasmids were sequenced at the Roswell Park Sequencing facility (Roswell Park Cancer Institute, Buffalo, NY).

Jamalzadeh S., & Cullen P.J. (2020). A Rab Escort Protein Regulates the MAPK Pathway That Controls Filamentous Growth in Yeast.

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Cloning Human KZNF cDNA Constructs

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Mammalian Gene Collection (MGC)-verified full length human KZNF cDNA clones were purchased from Thermo Fisher Scientific (Waltham, MA). PCR was performed to amplify ZNF cDNA using HotStar HiFidelity PCR kit (Qiagen, Valencia, CA). Primers were designed to amplify the region that includes KRAB and SCAN domains but not Zn finger motifs. Primers were also designed to include recognition sequences of the restriction enzymes as adaptors and three additional nucleotides to help binding of the restriction enzymes. The NCBI’s Primer-BLAST design tool was used to design primers and to calculate primer melting temperatures. After PCR, amplicons were digested with corresponding restriction enzymes to generate ligation-ready DNA fragments. For backbones, the pSG424 vector, which includes GAL4 DBD upstream of multiple cloning sites, was cut with the same restriction enzymes. Inserts were then ligated with the pSG424 backbone using Quick Ligation kit (New England BioLabs, Ipswich, MA). For transformation, 2µl of ligation reaction and One Shot MAX Efficiency DH5α-T1 competent cells (Invitrogen, Carlsbad, CA) were used according to the manufacturer’s protocol. Colonies were analyzed by restriction analysis and positive clones were sequenced (UW-Madison, Biotechnology Center) to verify correct incorporation and sequences of inserts.

Xiao T.Z., Suh Y, & Longley B.J. (2014). MAGE proteins regulate KRAB zinc finger transcription factors and KAP1 E3 ligase activity. Archives of biochemistry and biophysics, 563, 136-144.

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T Cell Receptor Sequencing Protocol

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Antigen-specific CD4⁺ T cell clones were stained as described above for viability and surface expression of CD3 and CD4 (reagent details in Key Resources Table). A maximum of 5 × 10³ live CD3⁺CD4⁺ T cells was sorted into 100 μL of RNAlater (Ambion) using a FACSAria III (BD Biosciences). Sorted cells were stored at −80°C. All expressed TRA and TRB gene transcripts were amplified using an unbiased template-switch anchored RT-PCR (Quigley et al., 2011 ). Pure mRNA was isolated from sorted cells using μMACS Oligo(dT) MicroBeads (Miltenyi Biotec), and cDNA was generated using a SmarterRACE Kit (Takara) with Superscript II (Invitrogen). Transcripts were amplified using an Advantage 2 PCR Kit (Takara) with the relevant constant region primers (reagent details in Key Resources Table). Amplicons were separated on agarose gels and extracted using a NucleoSpin Extract II Gel Extraction Kit (Macherey-Nagel). Subclones were generated using a TOPO TA Cloning Kit (Invitrogen) and One Shot Max Efficiency DH5α-T1 Competent Cells (Invitrogen). Conventional sequencing was performed as described previously (Price et al., 2005 (link)). Gene use was assigned using the ImMunoGeneTics (IMGT) nomenclature (Lefranc, 2003 (link)).

Brenna E., Davydov A.N., Ladell K., McLaren J.E., Bonaiuti P., Metsger M., Ramsden J.D., Gilbert S.C., Lambe T., Price D.A., Campion S.L., Chudakov D.M., Borrow P, & McMichael A.J. (2020). CD4+ T Follicular Helper Cells in Human Tonsils and Blood Are Clonally Convergent but Divergent from Non-Tfh CD4+ Cells. Cell reports, 30(1), 137-152.e5.

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T Cell Receptor Sequencing Protocol

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Efficient Site-Directed Mutagenesis Protocol

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For single-site directed mutagenesis, PCRs were run for 18 cycles of 15 s at 95 °C, 30 s at 60 °C, and 18 min at 72 °C using Q5 High-Fidelity DNA Polymerase (NEB M0491). In addition to GC enhancer, 3.5% DMSO was included in the PCR mix because of the GC-rich regions in the HIPK2 insert. The PCR products were treated with FastDigest DpnI (Thermo Scientific FD1704) to remove the template DNA, followed by transformation into One-Shot MAX Efficiency DH5α-T1 Competent Cells (Invitrogen 12297016). Multisite-directed mutagenesis was performed using the GeneArt Site-Directed Mutagenesis PLUS System (Invitrogen A14604) with slight modifications. DNA methylation was not included prior to PCR. Instead, template DNA was removed using DpnI digestion, followed by in vitro recombination reaction. See SI Appendix, Table S2 for the mutagenic primers used. Mutations were then verified by DNA sequencing.

Wong K.K., Liu T.W., Parker J.M., Sinclair D.A., Chen Y.Y., Khoo K.H., Vocadlo D.J, & Verheyen E.M. (2020). The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 117(4), 2004-2013.

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