cDNA encoding gp100, tyrosinase and TRP-2 was synthesized and cloned into pSP73-SphA64 (kindly provided by Professor E Gilboa, Duke University Medical Center, Durham, NC) using 5`XhoI/3`PacI restriction (Geneart/Life Technologies, Regensburg, Germany). The plasmids were propagated in E. coli competent cells (Invitrogen, Paisley, United Kingdom) and purified as described previously [36 (link)]. Prior to serving as a DNA template for in vitro transcription, all plasmids were linearized with SpeI restriction enzyme and purified using the Wizard DNA Clean-Up System (Promega, Oslo, Norway). The in vitro transcription was performed as previously described [36 (link)].
E coli competent cells
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
E. coli–competent cells are laboratory reagents designed for the transformation of DNA into E. coli bacterial cells. They facilitate the uptake of plasmid DNA, enabling genetic manipulation and recombinant protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Wizard dna clean up system, by Promega (1 mentions)
Bamhi, by Qiagen (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Pcr2.1 vector, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Human ifn γ elisa, by R&D Systems (1 mentions)
Human genomic dna, by Promega (1 mentions)
Fluostar omega multi mode microplate reader, by BMG Labtech (1 mentions)
Sybr green reaction mix, by Roche (1 mentions)
Lightcycler 480 sybr green, by Roche (1 mentions)
Chitin from shrimp shells, by Merck Group (1 mentions)
5 protocols using e coli competent cells
1
Synthesis and Cloning of Melanoma Antigens
2
Cloning SGK1 ARE I Regulatory Element
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The SGK1 ARE I was PCR-amplified using the primers listed in Supplemental Table 1 . The primers were designed such that they carried built-in restriction enzyme cut sites for BamHI and SbfI. The PCR product was then gel-purified (Qiagen, Cat# 28706) and cloned into the pCR™2.1 vector (Invitrogen, Cat# K2040-01), according to manufacturer's instructions. One shot®E.coli competent cells (Invitrogen, Cat# K2040-01) were used for transformation, and ampicillin (Sigma, Cat# A9393-5G) was used as the selectable marker. Restriction enzymes BamHI (New England Biolabs, Cat# R0136S) and SbfI (New England Biolabs, Cat# R0642S) were used to cut out the SGK1 ARE I regulatory element from the pCR™2.1 vector, which was gel purified and inserted into the the CpG-free lucia promoter vector (Invivogen, Cat# pcpgf-prom), using the same reaction conditions as described above. The vector was transformed into E.coli GT115 chemically competent cells (Invivogen, Cat# lyo-115-11) and E. coli were cultured overnight at 37°C on Fast-Media® Zeo agar (Invivogen, Cat# fas-zn-s).
3
Modulation of PBMC-E. coli DNA Responses
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Bacterial DNA was prepared from E. coli–competent cells (Thermo Fisher Scientific) using DNeasy Blood and Tissue Kit (Qiagen). PBMCs freshly isolated from each study participant (5 × 105 cells) were cocultured with E. coli bacterial DNA (0 to 3 × 104 DNA copies [7.5 pg/mL]). Coculture supernatants were collected and analyzed on day 3. IFN-γ levels in the supernatants were measured using a human IFN-γ ELISA (R&D Systems) and the FLUOstar Omega multimode microplate reader (BMG Labtech). For bacterial DNA digestion, E. coli bacterial DNA was incubated with DNase I (Thermo Fisher Scientific) at 37°C for 15 minutes. For BTK inhibition, PCI 29732 (final concentration 0.5 nM, Tocris Bioscience) (42 (link)) was added to PBMC cultures at 2, 8, and 12 hours prior to the addition of E. coli bacterial DNA (3 × 104 copies, 7.5 pg/mL). To examine potential interference by human cell-free DNA, an equivalent concentration (7.5 pg/mL) of human genomic DNA (Promega) was added to the bacterial DNA and PBMC coculture, and IFN-γ output was measured as described previously.
4
Quantitative PCR for 16S bacterial DNA detection
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Quantitative PCR was performed blinded for clinical characteristics. The amplification reaction mixture was composed of 10 μL of LightCycler 480 SYBR Green (Roche), 1 μL of 16S bacterial rDNA forward primer (5′-AAC AGG ATT AGA TAC CCT GGT AG-3′, 1 μL of reverse primer (5′-GGT TCT KCG CGT TGC WTC-3′) (72 (link)), 1 μL of dsDNAase (Thermo Fisher Scientific), 1 μL of 10× dsDNAase buffer (Thermo Fisher Scientific), and 5 μL of SYBR Green reaction mix (Roche). Then, 19 μL of the reaction mixture was added to each well of a 384-qPCR plate, followed by 1 μL of DNA isolated from 200 μL of serum (DNeasy Blood and Tissue Kit, Qiagen) or the E. coli bacterial DNA standard. The DNA was amplified in triplicate, and mean values were calculated. The reaction conditions for amplification of DNA were 95°C for 10 minutes, followed by 45 cycles at 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 5 seconds. The bacterial DNA standard was prepared from E. coli–competent cells (Thermo Fisher Scientific) using DNeasy Blood and Tissue Kit (Qiagen) and concentrations were measured by NanoDrop (Thermo Fisher Scientific). Using the E. coli genome length (4,700,000 bp), the number of E. coli DNA copies was calculated (https://cels.uri.edu/gsc/cndna.html ) and serial dilutions of this standard were used to define DNA copy numbers in a standard curve.
5
Bacterial Transformation with Chitin/Chitosan
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E. coli competent cells and the pET-21b(+)vector
were obtained from Thermo Fisher Scientific (Waltham, MA). Chitin
from shrimp shells with a da of 90% was purchased from Sigma-Aldrich
(St. Louis, MO), and chitosan with a da of 48% was a gift from Prof.
Finn Aachmann (NTNU, Norway). Chitosan with a da of 10% was obtained
from Mahtani Chitosan PVT Ltd (Gujarat, India). All other reagents
were of analytical grade unless otherwise stated.
were obtained from Thermo Fisher Scientific (Waltham, MA). Chitin
from shrimp shells with a da of 90% was purchased from Sigma-Aldrich
(St. Louis, MO), and chitosan with a da of 48% was a gift from Prof.
Finn Aachmann (NTNU, Norway). Chitosan with a da of 10% was obtained
from Mahtani Chitosan PVT Ltd (Gujarat, India). All other reagents
were of analytical grade unless otherwise stated.
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