pgMAX system-EFGP

Protein Expression Prokaryotic cells - E. coli EGFP

Experiment
Protein Expression Prokaryotic cells - E. coli EGFP
Product
pgMAX system-EFGP from Manabu Murakami, Department of Pharmacology, Hirosaki University
Manufacturer
Manabu Murakami, Department of Pharmacology, Hirosaki University

Protocol tips

Protocol tips
The PCR-amplified product was inserted into the EcoRV site of pgMAX and transfected in E. coli.

Publication protocol

"Bicistronic expression vectors, pIRESpuro3 and pZero-2, were purchased from Thermo Fisher Scientific. DsRed2 and pEGFP-N1 plasmids were purchased from Clontech Laboratories (Palo Alto, CA, USA) [9]. The Bgeo gene (a combined gene of β-galactosidase and neomycin phosphotransferase) was kindly provided by Dr. Smith [10]. For plasmid construction, PCR-based mutagenesis was performed. The conditions for PCR with high-fidelity Pfu DNA polymerase (Agilent Technologies) were empirically modified (denaturation at 94°C for 20 s, an annealing step at the calculated temperature (ca. 50°C) for 30 s and an extension at 72°C for 30 s, for 35 cycles). Amplified PCR products were gel-purified with a gel extraction kit (Macherey-Nagel GmbH, Dueren, Germany). The inhibitory unit (iUnit) was PCR-amplified from pZero2 (Invitrogen, Carlsbad, CA, USA)) with a specific oligo DNA (iFor and iRev, see S1 Table). Additional restriction enzyme sites were introduced with oligonucleotide DNA primers (S1 Table), and protocols using the pgMAX system as well as the entire pgMAX sequence have been deposited in protocols.io. (DOI dx.doi.org/10.17504/protocols.io.zq3f5yn). The SwaI restriction site; lac promoter (lacP) and operator (lacO); Kozak sequence (Kozak), PmeI restriction site; BamHI site; Flag protein tag; BamHI site; and EcoRI, EcoRV and XhoI restriction sites were constructed using a PCR. The blunt-end product was inserted into the EcoRV site in pBluescriptIISK- (Agilent Technologies) using standard ligation techniques (Takara DNA Ligation kit ver.2.1, Takara, Otsu, Japan). For the transformation, XL10-Gold ultracompetent cells (Tetr Δ(mcrA)183 Δ(mcrCB‒hsdSMR‒mrr)173 endA1 supE44 thi‒1 recA1 gyrA96 relA1 lac Hte [F'proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr] (Agilent Technologies) were used.

A blunt-end DsRed2 DNA fragment was amplified using Pfu DNA polymerase with DsRed2-specific oligo DNA (DsRed2for: AaaGCTAGCatgGCCTC CTCCGAGAAC GTCATCA; DsRed2rev: aaaGAATTCagatctcaggaacaggtggtg). A blunt-end enhanced green fluorescent protein (EGFP) DNA fragment was amplified using high-fidelity Pfu with EGFP-specific oligo DNA (EGFPfor: cccGCTAGCatgGTGAGCAAGGGCGAGGAG; EGFPrev: cccGGTACCGGCGGCGGTCACGAACTCCAG). The PCR-amplified product was inserted into the EcoRV site of pgMAX and transfected in E. coli."

Full paper   Login or join for free to view the full paper.

Reviews

pgMAX system-EFGP from Manabu Murakami, Department of Pharmacology, Hirosaki University has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli EGFP using pgMAX system-EFGP from Manabu Murakami, Department of Pharmacology, Hirosaki University.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Manabu Murakami, Department of Pharmacology, Hirosaki University for pgMAX system-EFGP below.

We haven't found the manufacturer protocol for this product yet.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli EGFP using pgMAX system-EFGP from Manabu Murakami, Department of Pharmacology, Hirosaki University. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms