Log In Sign up
The largest database of trusted experimental protocols

Prsetb

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States, Germany

The PRSETB is a lab equipment product from Thermo Fisher Scientific. It is a programmable and configurable system designed for laboratory applications. The core function of the PRSETB is to provide a versatile and customizable platform for various laboratory tasks, but a detailed description while maintaining an unbiased and factual approach is not available.

Automatically generated - may contain errors

Lab products found in correlation

Pcdna3, by Thermo Fisher Scientific (4 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Histone h3k9 methylation reporter, by Addgene (2 mentions) Qiaquick gel extraction kit, by Qiagen (2 mentions) Pfu turbo dna polymerase, by Agilent Technologies (1 mentions) Gibson assembly reagent, by New England Biolabs (1 mentions) Pyrobest polymerase, by Takara Bio (1 mentions) Petduet 1, by Merck Group (1 mentions) Prset b, by Merck Group (1 mentions) Pgex 2t, by GE Healthcare (1 mentions) Pgem t, by Promega (1 mentions) Histrap, by GE Healthcare (1 mentions) Complete protease inhibitor, by Roche (1 mentions) E coli dh5α strain, by Thermo Fisher Scientific (1 mentions) E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions) Model 422 electro eluter, by Bio-Rad (1 mentions) Pcdna3 mammalian expression vector, by Thermo Fisher Scientific (1 mentions) T4 dna ligase, by Thermo Fisher Scientific (1 mentions) Restriction endonuclease, by Thermo Fisher Scientific (1 mentions) E coli xl1 blue cells, by Thermo Fisher Scientific (1 mentions)

38 protocols using prsetb

1

Codon-Optimized Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
The LOC100003999 gene was codon optimized for E. coli and synthesized commercially (GeneScript USA Inc., Piscataway, NJ). The product was cloned into EcoRV site of pUC57-kan vector. The plasmid was digested with BglII and EcoRI and ligated into BamHI and EcoRI site of pRSET-B (Life Technologies, Carlsbad, CA) for the expression of N-terminal hexa-histidine-tagged protein. The zgc:113054 gene was also codon optimized for E. coli and commercially synthesized (GeneScript USA Inc.). The product was cloned into EcoRV site of pUC57-amp vector. The plasmid was digested with BglII and EcoRI and ligated into BamHI and EcoRI site of pRSET-B (Life Technologies) for the expression of N-terminal hexa-histidine-tagged protein.
Osborn A.R., Almabruk K.H., Holzwarth G., Asamizu S., LaDu J., Kean K.M., Karplus P.A., Tanguay R.L., Bakalinsky A.T, & Mahmud T. (2015). De novo synthesis of a sunscreen compound in vertebrates. eLife, 4, e05919.
+ Open protocol
+ Expand
2

Engineered Fluorescent Protein Variants

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
The CU17S gene was amplified using primers containing 5′-BamHI and 3′-XhoI sites, and the restricted product was cloned in-frame into the BamHI/XhoI sites of pRSETB (Thermo Fisher Scientific) to generate pRSETB/CU17S, which was used as the template for mutagenesis. Random mutations were introduced using error-prone PCR. Bacterial cells transformed with mutagenized plasmids were screened for efficient chromophore maturation at 37 °C. The obtained product with mutation V168A was pRSETB/StayGold.
On the other hand, EGFP, SiriusGFP, mClover3 and mNeonGreen genes were amplified using primers containing 5′-BamHI and 3′-EcoRI sites, and the restricted products were cloned in-frame into the BamHI/EcoRI sites of pRSETB to generate pRSETB/EGFP, pRSETB/SiriusGFP, pRSETB/mClover3 and pRSETB/mNeonGreen, respectively.
Likewise, mTFP1 (ref. 23 (link)), Venus45 (link), Achilles41 (link), mGold10 (link), mOrange2 (ref. 6 (link)), mCherry13 (link), mScarlet-I (ref. 7 (link)), mScarlet-H (ref. 7 (link)), mCardinal46 (link), TagRFP-T (ref. 6 (link)), AmCyan47 (link), tKeima48 (link), dKeima48 (link), KikG (ref. 49 (link)), h2-3 (ref. 50 ) and TurboRFP (ref. 51 (link)) genes were transferred to pRSETB vector using the BamHI and EcoRI sites.
Hirano M., Ando R., Shimozono S., Sugiyama M., Takeda N., Kurokawa H., Deguchi R., Endo K., Haga K., Takai-Todaka R., Inaura S., Matsumura Y., Hama H., Okada Y., Fujiwara T., Morimoto T., Katayama K, & Miyawaki A. (2022). A highly photostable and bright green fluorescent protein. Nature Biotechnology, 40(7), 1132-1142.
+ Open protocol
+ Expand
3

Construction of Chimeric Protein Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols
DNA fragments of the VldE-N-terminal domain, VldE-C-terminal domain, OtsA-N-terminal domain and OtsA-C-terminal domain were amplified by PCR using PfuTurbo DNA polymerase (Agilent Technology) and the listed primer pairs (Table S1). The 5´-end of the VldE-N-terminal domain DNA fragment was digested with XhoI and the 3´-end of the OtsA-C-terminal domain DNA fragment was digested with EcoRI. The resulted DNA fragments were ligated into the vector pRSET B (Invitrogen), predigested with XhoI and EcoRI, to give pRSET-B-chimera-1. Similarly, the 5´-end of OtsA-N-terminal domain DNA fragment and the 3´-end of VldE-C-terminal domain DNA fragment were digested with BamHI and EcoRI, respectively, and ligated into pRSET B, predigested with BamHI and EcoRI, to give pRSET-B-chimera-2. The identity of the chimera-1 and chimera-2 genes was confirmed by Sanger DNA sequencing in the Center for Genome Research and Biocomputing (CGRB) at Oregon State University.
Abuelizz H.A, & Mahmud T. (2015). Distinct Substrate Specificity and Catalytic Activity of the Pseudoglycosyltransferase VldE. Chemistry & biology, 22(6), 724-733.
+ Open protocol
+ Expand
4

Cell-Free Protein Synthesis from Engineered Antibody Fragments

Check if the same lab product or an alternative is used in the 5 most similar protocols
Continuously, from the previous DNA amplification steps, we prepared DNA fragments for CFPS and plasmids for transformation followed by sequence analysis.

Gibson assembly of the second PCR product and linearised pRSETb vector containing (i) the SKIK tag immediately after the start codon, (ii) fragments encoding Lc or Hc (IgM or IgG) fused with the leucine zipper A (LZA) for Hc and B (LZB) for Lc, and (iii) HA or His tags downstream of the LZ DNA (Fig. 1). The Gibson assembly reagent was purchased from New England Biolabs (Ipswich, MA). After mixing 1 µL of the second PCR reaction mixture, 1 µL of the linearised vector (100 ng), and 2 µL of 2 × Gibson Master Mix, they were incubated at 50 °C for 15 min. The genes in the vector were artificially synthesised for optimised codon usage for E. coli expression and embedded into pRSETb (Life Technologies, DNA sequences of linearized vectors are available in Supplementary Information). The linearised DNA fragments used here were amplified using Pyrobest™ Polymerase (Takara Bio). Purified DNA fragments were stored at −20 °C until use.

Preparation of DNA fragments for CFPS. DNA fragments containing the T7 promoter, gene (Lc or Hc), and T7 terminator were then amplified from the assembled products by PCR with Tsk Gflex polymerase. The amplified DNA fragments were directly used as the templates for the following CFPS step.

Ojima-Kato T., Nagai S, & Nakano H. (2017). Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation. Scientific Reports, 7, 13979.
+ Open protocol
+ Expand
5

Recombinant Expression and Purification of AgamOBP1

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
A PCR-amplified DNA fragment encoding AgamOBP1 (AF437884) was cloned into pRSET-B (Thermo Fisher Scientific, Waltham, MA, USA) and soluble recombinant protein (rAgamOBP1) was produced in E. coli BL21 Star (DE3) pLysS cells [18 (link),34 (link)]. The rAgamOBP1 protein was purified on a nickel-NTA column following the manufacturer’s directions (Thermo Fisher Scientific, Waltham, MA, USA), eluted with 5 mM EDTA and subjected to extensive dialysis against 50 mM Tris-HCl pH 7.4.
Dimitratos S.D., Hommel A.S., Konrad K.D., Simpson L.M., Wu-Woods J.J, & Woods D.F. (2019). Biosensors to Monitor Water Quality Utilizing Insect Odorant-Binding Proteins as Detector Elements. Biosensors, 9(2), 62.
+ Open protocol
+ Expand
6

Cloning and Expression of acb Cluster

Check if the same lab product or an alternative is used in the 5 most similar protocols
The genes acbU, acbJ, acbR, acbI, acbS and acbK were PCR amplified from gDNA of Actinoplanes sp. SE50/110 using their corresponding primers (Supplementary Table 3). The amplicons were digested with appropriate restriction enzymes (Supplementary Table 2) and ligated with pRSET-B (Thermo Fisher), pXY20147 (link), or pET28b (Novagen) vectors. All plasmids were listed in Supplementary Table 4. The plasmid pRSETB-valC was constructed by Minagawa et al.29 (link).
Tsunoda T., Samadi A., Burade S, & Mahmud T. (2022). Complete biosynthetic pathway to the antidiabetic drug acarbose. Nature Communications, 13, 3455.
+ Open protocol
+ Expand
7

Bicistronic Luciferase Expression in E. coli

Check if the same lab product or an alternative is used in the 5 most similar protocols
Primers used in this study were listed in Supplementary Table 1. The luxCDABE genes of Photorhabdus luminescens was cloned from pAKlux139 (link), which was provided by Attila Karsi (Addgene plasmid #14073). For bicistronic expression of luxAB operon in E. coli, PCR-amplified luxAB fragment was digested with BamHI and EcoRI, and the fragment was inserted in-frame into the corresponding site of pRSET B (Thermo Fisher Scientific). For the fusion protein expression, a 15-amino-acid linker (GGGGS)3 was inserted between luxA and luxB by overlap extension PCR as previously described34 . The resulting PCR product was inserted in-frame into the BamHI-EcoRI site of pRSET B.
For co-expression of two subunits of luciferase in E. coli by dual promoter from a single plasmid, the region including two multiple cloning sites (MCS) of pETDuet-1 (Merck) excised with BamHI and KpnI was inserted into the corresponding site of pRSET B. Venus and circularly permutated Venus series were cloned from BRAC derivatives19 . Venus or circularly permutated Venus variant was fused to a subunit of the luciferase by an EL (glutamic acid-leucine) linker encoded by SacI recognition sequence. The fusion constructs were inserted in-frame into the BamHI-NotI site of the MCS1. The PCR-amplified fragment of another subunit of luciferase was inserted in-frame into the NdeI-KpnI site of MCS2.
Kaku T., Sugiura K., Entani T., Osabe K, & Nagai T. (2021). Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Scientific Reports, 11, 14994.
+ Open protocol
+ Expand
8

Tandem Repeat Array Construction Using CTPRa

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The tandem repeat arrays of CTPRa or CTPR10D (a single CTPR with the ‘10D’ loop sequence) were built by concatemerization of individual CTPRa or CTPR10D using BamHI and BglII sites as previously described10 (link),29 (link). Briefly, a single consensus tetratricopeptide repeat (CTPRa1) was purchased as a short double-stranded DNA fragment and inserted into the T7-regulated expression vector pRSET B between the BamHI and HindIII restriction sites (ThermoFisher Scientific). The CTPRa1 fragment was then PCR-amplified using T7 promoter primers. The CTPRa1 PCR product and CTPRa1 pRSET B vector were then digested with BamHI/HindIII and BglII/HindIII restriction enzymes, respectively. The result is two concatamerized CTPRa1 genes froming a CTPRa2 (i.e. two-repeat array). The concatamerization of BamHI and BglII results in an Arg and a Ser after the highly conserved Pro31 of the CTPR sequence. As a result, the CTPRa2 contains the well-studied DPRS loop30 (link). This process can be repeated to generate CTPRa proteins of different lengths. Addition of the CTPR10D module generates the CTPRalt constructs. The sequences are given in Table S1.
Perez-Riba A., Komives E., Main E.R, & Itzhaki L.S. (2019). Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold. Scientific Reports, 9, 15439.
+ Open protocol
+ Expand
9

Mammalian Expression of 3X-FLAG-BAF60 Subunits

Check if the same lab product or an alternative is used in the 5 most similar protocols
Vectors for expression of 3X-FLAG-tagged BAF60 subunits in mammalian cells were previously described (Lores et al., 2010 (link)). Mitf deletion constructs were generated by PCR and sub-cloned into the Not1/MluI sites of a pCMV-V vector. For bacterial expression, BAF60A cDNA was subcloned into the Sal1/HindIII sites of pGEX-2T (GE Healthcare, Pittsburgh, PA, USA) and MITF cDNA was subcloned into the EcoRI/HindIII sites of pRSET-B (Thermofisher, Waltham, MA, USA). All constructs were verified by sequencing.
Aras S., Saladi S.V., Basuroy T., Marathe H.G., Lorès P, & de la Serna I.L. (2018). BAF60A mediates interactions between MITF and the BRG1-containing SWI/SNF complex during melanocyte differentiation. Journal of cellular physiology, 234(7), 11780-11791.
+ Open protocol
+ Expand
10

Engineered EphA4 Biosensors for Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols
The construct for the cytosolic EphA4 biosensor was generated by polymerase chain reaction (PCR) of the complementary DNA encoding the c-Src SH2 domain using a forward primer containing a SphI site and a reverse primer containing the gene sequence for a flexible linker (GSTSGSGKPGSGEGS), a substrate peptide containing the Y596 motif derived from the EphA4 juxtamembrane (JM) region and a SacI site. Similar biosensors including candidate substrates corresponding to Y602 from the EphA4 juxtamembrane region and Y610 from the EphB2 JM region were also generated. PCR products were fused with N-terminal enhanced CFP (ECFP) and C-terminal YPet. Y596F, R175V mutations were conducted using the Quickchange site-directed mutagenesis kit (Stratagene). Constructs were cloned using BamHI and EcoRI into the pRSETB plasmid (Invitrogen) for bacterial expression and into pcDNA3 for mammalian cell expression (Invitrogen).
The membrane-targeted Lyn-EphA4 biosensor was constructed by fusing 16 amino acids from the Lyn kinase (MGCIKSKRKDNLNDDE) to the N-terminus of the cytosolic EphA4 biosensor by PCR. The K-Ras-EphA4 biosensor was constructed by fusing the 14 amino acids of K-Ras-prenylation site (KKKKKKSKTKCVIM) to the C-termini of the cytosolic EphA4 biosensor by PCR. The PCR products for the Lyn- and K-Ras-EphA4 biosensors were subcloned into pcDNA3′ for mammalian cell expression.
Pan Y., Lu S., Lei L., Lamberto I., Wang Y., Pasquale E.B, & Wang Y. (2019). Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane. ACS sensors, 4(2), 294-300.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!